WO2021065665A1 - Method for producing three-dimensional cell structure - Google Patents

Method for producing three-dimensional cell structure Download PDF

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Publication number
WO2021065665A1
WO2021065665A1 PCT/JP2020/035998 JP2020035998W WO2021065665A1 WO 2021065665 A1 WO2021065665 A1 WO 2021065665A1 JP 2020035998 W JP2020035998 W JP 2020035998W WO 2021065665 A1 WO2021065665 A1 WO 2021065665A1
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cell
cells
template
cell population
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PCT/JP2020/035998
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French (fr)
Japanese (ja)
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敬太 山下
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株式会社カネカ
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a method for producing a three-dimensional cell structure.
  • Non-Patent Document 1 Non-Patent Document 1
  • Patent Document 1 discloses a method in which cell particles are put into a regulation frame portion to form a flat tissue and then press-molded. By applying an external stimulus called external pressure, a cell structure with high strength and biocompatibility can be formed. Further, a cell structure having a desired shape can be obtained by providing a groove portion, a through hole, a curved shape in a bent shape, or the like in the press molding portion.
  • Patent Document 2 discloses a method of molding a tubular structure using a silicon template using a cell structure containing a polymer (mosaic cell mass) as a raw material. There are four holes in the core holder of the lower base to improve the liquid diffusivity and improve the survival of cells.
  • Patent Document 3 discloses a method for obtaining a cell structure having a desired shape by culturing a cell mass after penetrating a cell mass through a ring-shaped sword ridge in order to form the cell into a tubular shape. ing.
  • Non-Patent Document 2 discloses a method of molding a single cell as a raw material using a mold made of agarose gel.
  • the method for producing a three-dimensional cell structure disclosed in the above prior art document has the following problems.
  • Patent Document 1 The mold used by the method described in Patent Document 1 is flat because it is pressed and molded, and does not have a mechanism for increasing the diffusivity of the culture solution. Therefore, there is a problem that a structure having an arbitrary shape cannot be easily manufactured. In addition, there is also a problem that the nutrient supply to the inside of the tissue tends to be insufficient because the diffusivity of the culture solution is low. In addition, applying external stimuli to cells by press molding carries the risk of adversely affecting tissue function.
  • a tubular structure can be produced as a three-dimensional cell structure.
  • a structure having an arbitrary shape cannot be manufactured.
  • the nutrient supply to the inside of the tubular three-dimensional cell structure is still insufficient only by the drainage holes provided in the mold.
  • Patent Document 3 has a problem that the productivity is extremely low because it is necessary to individually fix the cell mass to the tubular sword ridge. Further, the shape of the three-dimensional cell structure is limited to a simple one, and there is also a problem that the cell density of the obtained three-dimensional cell structure is low and the shape tends to be non-uniform. Furthermore, since the cell mass is pierced and fixed with a sword mountain, there is a problem that the physical load on the cell is large and cell death is easily induced.
  • Non-Patent Document 2 Since the method described in Non-Patent Document 2 uses a single cell as a raw material, the obtained three-dimensional cell structure tends to have a low cell density and a non-uniform shape, and there is a problem in the performance and yield of the cell structure.
  • a method for producing a three-dimensional cell structure wherein a cell population arranging step of arranging a cell population containing a cell mass having an average diameter of 50 ⁇ m or more and 750 ⁇ m or less in the internal space of a template, and the cell population are described.
  • the production method comprising a culturing step of culturing in a medium together with the mold, wherein the wall portion of the mold has a porous structure through which liquid can pass.
  • the production method according to (1) or (2), wherein the internal space of the mold is cylindrical.
  • a highly uniform cell structure having an arbitrary three-dimensional shape can be easily produced.
  • a three-dimensional cell structure having high shape uniformity and high physical strength can be stably produced.
  • FIG. A is a diagram immediately after the cell population placement step
  • B is a diagram on the 7th day of culturing.
  • the arrows indicate the arranged cell population (mainly the cell mass).
  • FIG. A is a diagram immediately after the cell population placement step
  • B is a diagram on the 7th day of culturing.
  • the arrows indicate the arranged cell population (mainly the cell mass). It is a figure which showed the result of observing the state of the cell culture in the culture step after the cell population arrangement step of the comparative example 1.
  • FIG. A is a diagram immediately after the cell population placement step
  • B is a diagram on the 7th day of culturing.
  • the arrows indicate the arranged cell population (mainly the cell mass). It is a figure which showed the result of having confirmed the state of the structure (arrow) at the time of removing the mold of Example 2.
  • the arrowhead indicates a stainless steel cylinder installed as an inner wall in the space inside the mold.
  • FIG. 1 It is a figure which showed the state of the three-dimensional cell structure after removal of the template and the inner wall part of Example 2.
  • FIG. It is a figure which showed the state of the tissue (arrow) at the time of removing the mold of the comparative example 1.
  • FIG. In the figure, the arrowhead is a stainless steel cylinder installed as an inner wall in the space inside the mold.
  • the first aspect of the present invention is a method for producing a three-dimensional cell structure.
  • a cell population containing a cell mass having an average diameter in a specific range is placed inside a mold having a wall surface having a porous structure with fluid permeability.
  • cell clusters can be arranged at high density in the internal space.
  • a cell structure having a high cell density having an arbitrary three-dimensional shape can be easily produced.
  • Three-dimensional cell structure refers to a three-dimensional structure composed of a cell population. Therefore, it is composed of a cell layer such as a cell sheet and does not include a planar structure having a substantially uniform thickness.
  • the three-dimensional cell structure may be composed only of cells, but may contain substances other than cells. Examples of substances other than cells include, but are not limited to, extracellular matrix, a part of a template, and the like.
  • the three-dimensional cell structure may have a scaffold or may be scaffold-free. It is preferably scaffold-free.
  • scaffold means a scaffold for growing cells.
  • the scaffold is preferably composed of a material that is absorbed by a living body, and examples of such a material include, but are not limited to, polylactic acid, collagen, and the like.
  • a material that is absorbed by a living body examples include, but are not limited to, polylactic acid, collagen, and the like.
  • Each cell constituting the three-dimensional cell structure may be of the same or different types.
  • the shape of the three-dimensional cell structure may be either a fixed shape or an indefinite shape.
  • Examples of the standard shape include a spherical shape, an elliptical shape, a cubic shape, a rectangular shape, a polyhedral shape, a conical shape, a pyramidal shape, a cylindrical shape, a prismatic shape, a spiral shape, a donut shape shape, and a tubular shape. Not limited to.
  • Cell adhesion in the present specification is a constituent unit of a three-dimensional cell structure, and means an adhesive cell capable of cell-cell adhesion or cell-extracellular matrix adhesion unless otherwise specified. .. Adhesion of adherent cells to the cell culture substrate, cells, or extracellular matrix is referred to as "cell adhesion".
  • the type of cell in the present specification is not limited.
  • cells constituting a living tissue cells derived from cells constituting a living tissue, stem cells, or cells differentiated from stem cells can be mentioned.
  • the cell in this specification may be a cell isolated from a living tissue.
  • the "living tissue” refers to various tissues constituting the living body of an organism.
  • epithelial tissue, endothelial tissue, connective tissue, muscle tissue, nerve tissue and the like can be mentioned.
  • cells constituting these tissues include epithelial cells, endothelial cells, fibroblasts, muscle cells, nerve cells, and chondrocytes.
  • Stem cell refers to a cell that has the ability to differentiate into various cells and the ability to self-renew. For example, adult stem cells, pluripotent stem cells and the like can be mentioned.
  • an “adult stem cell” is a stem cell that exists in each adult tissue, has not completed final differentiation, and has a certain degree of pluripotency, and is a somatic stem cell or tissue. It is also called a sex stem cell. Examples thereof include neural stem cells, intestinal epithelial stem cells, hematopoietic stem cells, hair follicle stem cells, pigment stem cells, mesenchymal stem cells and the like.
  • Neurological stem cells are mainly nerve cells and cells having the ability to differentiate into glial cells.
  • Intestinal epithelial stem cells are cells having the ability to differentiate into epithelial cells that mainly constitute the inner wall of the digestive tract such as the small intestine and the large intestine.
  • “Hematopoietic stem cells” are cells having the ability to differentiate into blood cells such as erythrocytes, leukocytes, and platelets.
  • Hair follicle stem cells are cells having the ability to differentiate into hair follicle epithelial cells such as hair follicle stem cells and hair root sheath cells, as well as fat gland cells and basal cells.
  • chromophore stem cell is a cell having an ability to differentiate into a chromatophore mainly.
  • the adult stem cells used in the present specification commercially available cells or cells that have been distributed may be used, or newly prepared cells may be used.
  • MSCs Mesenchymal stem cells
  • CD73 5-Nucleotidase
  • CD90 Thy-1
  • CD45 PPRC (Protein tyrosine phosphatase, receptor type, C)
  • Mesenchymal stem cells may have the ability to differentiate into cells belonging to the mesenchymal system such as osteoblasts, adipocytes, muscle cells, and chondrocytes.
  • Mesenchymal stem cells can be collected from, for example, bone marrow, fat, fetal appendages, pulp and the like.
  • the "fetal appendage” is an organ necessary for the foetation to develop in the uterus, and examples thereof include placenta, umbilical cord, amniotic membrane, egg membrane, chorion, decidua, and amniotic fluid.
  • the mesenchymal stem cells used herein are mesenchymal stem cells that have been genetically modified or have not been genetically modified, and are preferably non-genetically modified mesenchymal stem cells.
  • the ratio of cells positive for surface antigen indicates the ratio of cells positive for surface antigen analyzed by flow cytometry.
  • the ratio of cells in which the surface antigen is positive may be described as “positive rate”.
  • the “ratio of cells in which the surface antigen is negative” in the present specification indicates the ratio of cells in which the surface antigen is negative in the surface antigen analyzed by flow cytometry.
  • the ratio of cells in which the surface antigen is negative may be described as "negative rate”.
  • a “pluripotent stem cell” is a pluripotent (pluripotent) cell capable of differentiating into all types of cells constituting a living body, and is cultured in vitro under appropriate conditions. A cell that can continue to grow indefinitely while maintaining pluripotency.
  • embryonic stem cells ES cells: embryonic stem cells
  • EG cells embryonic germ stem cells
  • GS cells germline stem cells
  • iPS cells induced pluripotent stem cells
  • Pluripotent stem cells Pluripotent stem cells
  • EG cells are pluripotent stem cells prepared from fetal primordial germ cells (Shamblott MJ et al., 1998, Proc. Natl. Acad. Sci. USA., 95: 13726-13731. ).
  • GS cells are pluripotent stem cells prepared from the cell testis (Conrad S., 2008, Nature, 456: 344-349).
  • the "iPS cell” refers to a pluripotent stem cell that can be reprogrammed to bring the somatic cell into an undifferentiated state by introducing a gene encoding a small number of reprogramming factors into the differentiated somatic cell. ..
  • pluripotent stem cell used in the present specification, a commercially available cell or a cell for sale may be used, or a newly prepared cell may be used. Although not limited, iPS cells or ES cells are preferable as the pluripotent stem cells when used in each invention of the present specification.
  • iPS cells used in the present specification are commercially available products, for example, 253G1 strain, 201B6 strain, 201B7 strain, 409B2 strain, 454E2 strain, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS -RIKEN-12A strain, Nippons-B2 strain, TkDN4-M strain, TkDA3-1 strain, TkDA3-2 strain, TkDA3-4 strain, TkDA3-5 strain, TkDA3-9 strain, TkDA3-20 strain, hiPSC38-2 A strain, MSC-iPSC1 strain, BJ-iPSC1 strain and the like can be used. In addition, newly prepared clinical grade iPS cells may be used.
  • the combination of genes of the reprogramming factors to be introduced is not limited, but is, for example, OCT3 / 4 gene, KLF4 gene, SOX2 gene and c. -Myc gene combination (YuJ, et al. 2007, Science, 318: 1917-20.), OCT3 / 4 gene, SOX2 gene, LIN28 gene and Nanog gene combination (Takahashi K, et al. 2007, Cell, 131: 861-72.) Can be used.
  • the form of introduction of these factors into cells is not particularly limited, and examples thereof include gene transfer using a plasmid, introduction of synthetic RNA, and direct introduction as a protein.
  • iPS cells prepared by a method using microRNA, RNA, low molecular weight compounds and the like may be used.
  • newly prepared clinical grade iPS cells may be used.
  • KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SEES1 strain, and SEES2 strain are not limited.
  • SEES3 strain, SES-4 strain, SES-5 strain, SES-6 strain, SES-7 strain, HUES8 strain, CyT49 strain, H1 strain, H9 strain, HS-181 strain and the like can be used.
  • the cell-derived species in the present specification may be a multicellular organism. It is preferably an animal, more preferably a mammal.
  • rodents such as mice, rats, hamsters, guinea pigs, gerbils, livestock or pets such as dogs, cats, rabbits, cows, horses, pigs, sheep, goats, ferrets, and humans, crab monkeys, red-tailed monkeys, common marmosets. , Japanese monkeys, gorillas, chimpanzees and other primates.
  • human preferably human.
  • the cells herein may be autologous, allogeneic or heterologous cells, preferably allogeneic cells.
  • the cells herein may be cultured cells or passaged cells.
  • Cell mass is a mass-like aggregate formed by cell aggregation. Also called cell agglutinin or spheroid.
  • Cell aggregation means that a plurality of cells are three-dimensionally aggregated to form an agglutination. There are agglutination by different cells and agglutination by the same cell, whichever is used herein. Aggregation by the same cell includes the case where agglomerates are formed by the proliferation of one cell.
  • the mechanism of cell aggregation includes, but is not limited to, adhesion between cells via membrane proteins, cell membranes, extracellular matrix, and adhesion between cells via cadherin on the cell surface.
  • the shape of the cell mass is not particularly limited. Since the cell mass formed by cell aggregation usually has a substantially spherical shape, a substantially spherical shape is preferable.
  • the size of the cell mass is 50 ⁇ m or more, 100 ⁇ m or more, 150 ⁇ m or more, 200 ⁇ m or more, 220 ⁇ m or more, 250 ⁇ m or more, 300 ⁇ m or more, 350 ⁇ m or more, or 400 ⁇ m or more, and 750 ⁇ m or less, 700 ⁇ m or less, 650 ⁇ m or less, 600 ⁇ m.
  • it is 550 ⁇ m or less, 500 ⁇ m or less, or 450 ⁇ m or less.
  • the “diameter” in the present specification means the diameter ( ⁇ m) of a substantially spherical cell mass
  • the “average diameter” in the present specification is an arithmetic mean ( ⁇ m) of the diameters of a plurality of cell clusters. ) Means.
  • the measurement of the average diameter of the cell mass can be calculated from an image obtained by photographing the cell mass using, for example, a microscope and imaging software (for example, cellSense manufactured by OLYMPUS).
  • the average diameter of the cell mass can be obtained by measuring the diameter from any three directions of the photographed image of the cell mass and calculating the average value thereof.
  • the average diameter of the cell mass is within the above range, it is possible to reduce and homogenize the gap between the contact portions between the cell masses that may occur when the cell mass is placed in the inner space of the template, and as a result. , It is possible to prevent the occurrence of cracks in the cell culture in the culture step. Therefore, the cell density and shape uniformity of the obtained three-dimensional cell structure can be improved. If the average diameter of the cell mass is less than 50 ⁇ m, the shape of the obtained three-dimensional cell structure becomes non-uniform, which is not preferable.
  • the gap between the contact portions between the cell masses in the inner space of the template becomes large, so that the cell masses are unevenly fused in the culture step, resulting in cracks and a rough surface. It is not preferable because a cell culture having the above is formed.
  • the term "cell population” refers to a population consisting of a plurality of cells including at least the cell mass.
  • the cell population may consist solely of cell clusters or may include a single cell or several cells.
  • Each cell mass and / or each cell constituting the cell population can exist separately from each other in a liquid such as a culture solution without cell adhesion in the cell population.
  • the liquid containing the cell population in this case is often referred to herein as a "cell suspension.”
  • the ratio of cell clusters within the above size range is 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more. , 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more.
  • the cell population contains 50% or more of the cell mass
  • the homogeneity of the cell population is enhanced, and as a result, the arrangement of the cell mass in the inner space of the template becomes more uniform, and the cell mass fuses more uniformly in the culture step. Therefore, it is possible to obtain a three-dimensional cell structure having no visible cracks and further improved shape uniformity.
  • the cell population of the present invention can contain any number of cell clusters of 2 or more, eg, 1.0 ⁇ 10 1 , 1.0 ⁇ 10 2 , 2.0 ⁇ 10 2 , 5, .0 ⁇ 10 2 pcs, 1.0 ⁇ 10 3 pcs, 2.0 ⁇ 10 3 pcs, 5.0 ⁇ 10 3 pcs, 1.0 ⁇ 10 4 pcs, 2.0 ⁇ 10 4 pcs, 5.0 ⁇ 10 4 pieces, 1.0 ⁇ 10 5 pieces, 1.0 ⁇ 10 6 pieces, 1.0 ⁇ 10 7 pieces, 1.0 ⁇ 10 8 pieces, 1.0 ⁇ 10 9 pieces, 1.0 ⁇ 10 It can include, but is not limited to, 10 or more or less cell clusters.
  • the plurality of cell clusters or the plurality of cells contained in the cell population may be derived from the same species or different species from each other.
  • the cell suspension used in the present specification may contain any component other than cells, and examples thereof include salts, sugars, proteins, amino acids, medium components, buffers, and the like. Not limited.
  • the pH of the cell suspension can be near neutral, for example, pH 5.5 or higher, pH 6.0 or higher, pH 6.5 or higher or pH 7.0 or higher, and pH 10.5 or lower, pH 9 or higher. It can be 5.5 or less, pH 8.5 or less, or pH 8.0 or less, but is not limited thereto.
  • medium refers to a liquid, semi-solid or solid substance prepared for culturing cells. As a general rule, it contains more than the minimum necessary components essential for cell proliferation and / or maintenance. Unless otherwise specified, the medium used in the present specification corresponds to a liquid medium for animal cells used for culturing animal-derived cells. In addition, the medium is not limited, but is BME medium, BGJb medium, CMRL1066 medium, Glasgo MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscover's Modified Dulvecco'S Medium), Medium Medium 199 medium.
  • DMEM medium Dulvecco'S Modified Eagle'S Medium
  • ham F10 medium for example, ham F12 medium (Dulvecco'S Medium)
  • ham F12 medium for example, RPMI 1640 medium
  • Fisher'S medium for example, DMEM / F12 medium (Dulvecco'S Medium)
  • Eagle'S Medium / Nutrition Mixture F-12 Ham) can be used.
  • the weight ratio of the DMEM medium and the ham F12 medium is preferably in the range of 60/40 or more and 40/60 or less, for example, 58/42, 55/45, 52/48, 50/50, 48 /.
  • a medium mixed with 52, 45/55, 42/58, etc. is used.
  • the medium used for culturing human iPS cells and human ES cells can also be preferably used.
  • the medium used in the present invention may be a medium containing serum or a serum-free medium.
  • Serum is, but is not limited to, fetal bovine serum (FBS), horse serum and the like.
  • FBS fetal bovine serum
  • the final concentration of serum in the medium is 1% or more, 2% or more, 3% or more, 4% or more or 5% or more, and 20% or less, 18% or less, 16% or less. , 14% or less, 12% or less or 10% or less, more preferably 9% or less, still more preferably 8% or less.
  • the serum-free medium is not limited, but is, for example, STK1 or STK2 (manufactured by DS Pharma Biomedical), EXPREP MSC Medium (manufactured by Biomimetic Sympathies), Corning stemgro human adhesive stem cell medium (manufactured by Corning), etc. It may be a commercially available serum-free medium.
  • the medium used in the present specification can be prepared as a medium specific to various cells by adding various culture additives.
  • a "culture additive” is a substance added to a medium for the purpose of culturing.
  • Specific examples of culture additives include, but are not limited to, serum, serum substitute reagents, insulin, transferrin, selenium, cytokines, growth factors, albumin, sodium hydrogen carbonate, fatty acids, amino acids (eg, non-essential amino acids), L-ascorbin. Examples include acids, vitamins, antioxidants, 2-mercaptoethanol, pyruvate, buffers, inorganic salts, polysaccharides, antibiotics and the like.
  • Insulin, transferrin, and cytokines may be of natural origin isolated from animal (preferably human, mouse, rat, bovine, horse, goat, etc.) tissues or sera, or genetically engineered. It may be a recombinant protein.
  • the growth factors are not limited, but are, for example, FGF2 (Basic fiberblast growth factor-2), EGF (Epidermal growth factor), TGF- ⁇ 1 (Transforming growth factor- ⁇ 1), VEGF (VEGF). (Platelet-developed growth factor), Activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF and IGFBP-7 can be used.
  • Antibiotics include, but are not limited to, penicillin, streptomycin, amphotericin B and the like.
  • a particularly preferred growth factor as a culture additive for the medium used in the present invention is FGF2.
  • One or more culture additives can be contained in the medium.
  • Culture additives can be added to the medium in the form of solutions, derivatives, salts, mixed reagents and the like.
  • L-ascorbic acid may be added to the medium in the form of a derivative such as magnesium 2-ascorbic acid
  • selenium may be added to the medium in the form of a selenium salt (sodium selenite, etc.). May be good.
  • Insulin, transferrin, and selenium can also be added to the medium in the form of an ITS reagent (insulin-transferrin-selenium). It is also possible to use a commercially available medium to which at least one selected from L-ascorbic acid, insulin, transferrin, selenium and sodium hydrogen carbonate has already been added.
  • CHO-S-SFM II Life Technologies Japan Co., Ltd.
  • Hybridoma-SFM Life Technologies Japan Co., Ltd.
  • eRDF Dry Powered Media Life Technologies Japan Co., Ltd.
  • UltraCULTURE TM (BioWittaker), UltraDOMA TM (BioWhittaker), UltraCHO TM (BioWhittaker), UltraMDCK TM (BioWhiteker), STEMPRO (Registered Trademarks) (Veritas) and the like.
  • the term "template” refers to a molding mold for producing a three-dimensional cell structure. Due to its nature, the template has an internal space (cavity: sometimes referred to as “internal space” in the present specification), and the internal space is usually the appearance of a three-dimensional cell structure. It has a shape that exhibits.
  • the outer shape of the mold is not particularly limited. It may have a shape similar to or dissimilar to the three-dimensional cell structure to be produced.
  • the shape of the internal space has a shape similar to the external shape of the three-dimensional cell structure to be manufactured, in whole or in part. Therefore, the shape of the internal space can be made into a desired shape according to the external shape of the three-dimensional cell structure. For example, if it is desired to produce a cylindrical three-dimensional cell structure, the internal space of the template may be cylindrical.
  • the wall portion of the mold is thin, the outer shape and the shape of the inner space are similar. Therefore, it approaches the similar shape of the three-dimensional cell structure that also manufactures the outer shape.
  • the mold can have an inner wall portion (core) arranged in the internal space.
  • the internal space corresponds to a concave portion (female type) and the inner wall portion corresponds to a convex portion (male type), and the hollow portion formed between the internal space and the inner wall portion is the target three-dimensional cell. It exhibits the shape of a structure.
  • the shape of the inner wall portion can be a desired shape according to the outer or inner shape of the three-dimensional cell structure.
  • the internal space of the template may be cylindrical and the inner wall may be cylindrical, which is smaller than the internal space.
  • a donut-shaped or tubular three-dimensional cell structure can be produced by adjusting the amount of the cell population to be filled in the donut-shaped or tubular cavity formed between the internal space and the inner wall portion.
  • the material of the mold may be any material as long as it can have a porous structure through which all or part of the wall can pass, and is not particularly limited. Since the template is immersed in the medium as described later, it is desirable that the template is a water-insoluble material or a non-soluble material.
  • Water-insoluble material means a material that is insoluble in water or an aqueous solution. Includes inanimate systems, biological systems, and mixtures thereof. An inanimate water-insoluble material is a water-insoluble material whose material is not directly derived from a living organism. For example, plastic (including chemical fibers), glass, metal, silicon, synthetic rubber, ceramics and the like can be mentioned.
  • plastics for example, polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), polyurethane (PU), polyvinylidene chloride (PVC), polyvinylidene chloride (PVDC), polycarbonate (PC). ), Polysulfone (PSU), polyarylate (PAR), polyamide (nylon), polyvinyl alcohol (PVA) and the like can be used. If it is a metal, for example, a pure metal such as gold (Au), platinum (Pt), silver (Ag), copper (Cu), nickel (Ni), titanium (Ti), aluminum (Al), tartar (Ta), etc.
  • a pure metal such as gold (Au), platinum (Pt), silver (Ag), copper (Cu), nickel (Ni), titanium (Ti), aluminum (Al), tartar (Ta), etc.
  • Biological water-insoluble materials refer to water-insoluble materials produced directly by living organisms. Examples thereof include natural fibers (cellulose, keratin, fibroin, etc.), natural resins (eg, natural rubber or lacquer), bones (including coral skeleton, spongy skeleton, etc.), teeth, horns, xylem, charcoal, and the like.
  • the "non-soluble material” means a material that does not dissolve in water or an aqueous solution under normal temperature and pressure. Although not limited, it is usually of biological origin. Examples include polysaccharide macromolecules (eg, agar, mannan), gelled proteins (eg, gelatin, collagen), or mixtures thereof.
  • the mold may be a composite material composed of two or more different materials.
  • a material facing the outside of the mold and a material facing the internal space may be different materials.
  • the wall surface of the interior space is a cell contact surface that comes into direct contact with cells.
  • the inner space wall surface is preferably made of a non-cell adhesive material. This is because when the cell contact surface is a material having high cell adhesion, it becomes difficult to separate the three-dimensional cell structure and the template after production.
  • the cell non-adhesive material include stainless steel, aluminum, polypropylene and the like.
  • a material having a surface coated with a substance that inhibits cell adhesion can be adopted as the internal space wall surface.
  • the substance that inhibits cell adhesion include, but are not limited to, agar, poly (2-hydroxyethyl methacrylate), polyethylene glycol, and the like.
  • the mold used in the present invention has a porous structure that allows liquid to pass through all or part of the wall portion as described above.
  • “Wall portion” refers to a portion of the mold that constitutes the wall surface (including the side surface, bottom surface, and top surface) of the internal space.
  • liquid passable means that the liquid can pass through the wall portion. Due to this liquid permeability in the wall part, the liquid can freely move between the outside and the inside space of the part.
  • the "liquid” referred to here is mainly water or an aqueous solution, although it is not limited.
  • Aqueous solution includes medium and buffer.
  • As the buffer a darbecolinic acid buffer (DPBS), an Earl balanced salt solution (EBSS), a Hanks balanced salt solution (HBSS), a phosphate buffer (PBS) and the like can be used, but the buffer is not limited thereto.
  • the "porous structure” means a structure having a plurality of pores. All or part of the holes are open on the surface of the material having a porous structure.
  • the porous structure include a mesh structure and a structure in which a bubble structure has a perforated surface.
  • the shape of the pores in the porous structure is not limited. It may be circular, substantially circular, elliptical, polygonal, substantially polygonal, amorphous, or a combination thereof. It is preferable that the upper limit of the individual pore size is a size that the cell mass cannot pass through, and that the cell population can be supported when the cell population is laminated in the template internal space and the template can be self-supporting.
  • the lower limit is preferably a size capable of maintaining liquid permeability without causing clogging of the holes.
  • D the ratio of the major axis and / or the maximum diagonal of the pore to the average diameter of the cell mass (d /).
  • D) is greater than 1.0, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, or 2.0 or more, and 3.0 or less, 2. It may be 9 or less, 2.8 or less, 2.9 or less, 2.6 or less, or 2.5 or less.
  • the ratio (d / D) is 1.0 or less, the cell mass arranged in the mold internal space may pass through the pores, which is not preferable. Further, if the ratio (d / D) is larger than 3.0, when the cell mass is arranged in the template internal space, the pores may be clogged by the cell mass, and sufficient liquid permeability may not be ensured. , Not preferable.
  • the major axis and / or the maximum diagonal of the hole is 50 ⁇ m or more, 100 ⁇ m or more, 150 ⁇ m or more, 200 ⁇ m or more, 220 ⁇ m or more, 250 ⁇ m or more, or 300 ⁇ m or more, and 750 ⁇ m or less, 700 ⁇ m or less, 650 ⁇ m or less, It is 600 ⁇ m or less, 550 ⁇ m or less, 500 ⁇ m or less, or 450 ⁇ m or less.
  • the pore opening ratio in the porous structure region of the mold wall portion means the ratio of the pore portion to the total area. For example, in the case of a mesh structure or the like, when the average diameter of the hole portion is “A” and the wire diameter is “a”, it becomes (A / A + a) 2 ⁇ 100 (%).
  • the aperture ratio in the present specification is 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more, and 90% or less, 85% or less, It is preferably 80% or less, or 75% or less.
  • the aperture ratio By setting the aperture ratio to 40% or more and 90% or less, it is possible to secure the nutrient supply and support for the cell population in the internal space, and to form a three-dimensional cell structure while maintaining the viable state. If the aperture ratio is less than 40%, sufficient fluid permeability to the internal space cannot be maintained during static culture, and the nutrient supply to the cell population in the internal space cannot be secured, which is not preferable. Further, if the aperture ratio is larger than 90%, the template strength for supporting the cell population in the internal space may be insufficient, which is not preferable.
  • the material and characteristics of the inner wall portion described above may be the same as those of the mold, or may be completely or partially different.
  • the cell adhesion surface of the inner wall portion is preferably a cell non-adhesion material as in the template.
  • the inner wall portion may not have liquid permeability, or may have a porous structure having liquid permeability similar to the mold. When it has liquid permeability, the specific porous structure may be the same as or different from that of the mold. For example, there is a case where the side surface of the mold wall portion has a mesh structure and the inner wall portion has a porous structure having a bubble structure.
  • the template having the above structure can also be used as a template capable of embodying the method for producing a three-dimensional cell structure of the present invention, that is, a template for producing a three-dimensional cell structure.
  • the flow of the method for producing a three-dimensional cell structure of the present invention is shown in FIG.
  • the production method of the present invention includes a cell population arrangement step (S0102) and a culture step (S0103) as essential steps, and a cell population preparation step (S0101) and a separation step (S0104) as selection steps.
  • S0102 cell population arrangement step
  • S0103 culture step
  • S0101 cell population preparation step
  • S0104 separation step
  • the “cell population preparation step” (S0101) is a step of preparing a cell population containing a cell mass having an average diameter of 50 ⁇ m or more and 750 ⁇ m or less. This step is a selective step carried out prior to the cell population placement step.
  • the method for preparing the cell population is not particularly limited. Basically, it may be prepared so that a cell mass having a predetermined size is contained in the cell suspension based on a method for preparing a cell suspension known in the art.
  • a method for preparing a cell suspension containing a cell mass for example, a cell obtained by an adhesive culture method or a tissue piece collected from a living body or the like is used, for example, a Prime Surface (registered trademark) plate (manufactured by Sumitomo Bakelite Co., Ltd.) or the like.
  • a method of culturing on a cell non-contact substrate There is a method of culturing on a cell non-contact substrate.
  • the cells can spontaneously aggregate and form a cell mass.
  • Other methods of preparing cell suspensions containing cell clusters include hanging drop methods, chemical treatments and / or methods in which the droplets of the cell suspension are maintained in a suspended form and the cells aggregate in the droplets. There are a method of preparing by physical treatment, a method of preparing by suspension culture method, and the like.
  • the "adhesive culture method” refers to a culture method in which cells are adhered to a culture vessel or the like and, in principle, are grown in a single layer.
  • the “chemical treatment” refers to chemically treating monolayer cells and tissue pieces obtained by the adhesion culture method with a cell release agent used for detachment of adherent cells and cell dispersion of tissues.
  • the cell exfoliating agent trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA) and the like can be used, but the cell exfoliating agent is not particularly limited.
  • EDTA ethylenediaminetetraacetic acid
  • trypsin-EDTA solution manufactured by Thermo Fisher Scientific
  • TrypLE Select manufactured by Thermo Fisher Scientific
  • Accutase manufactured by Thermo Fisher Scientific
  • Accutase manufactured by Thermo Fisher Scientific
  • Accutase manufactured by Thermo Fisher Scientific
  • Accutase manufactured by Stemcell Technology
  • "physical treatment” promotes the separation of cell adhesion by applying a mechanical external force such as peeling treatment or suspension treatment to monophasic cells or tissue pieces whose cell adhesion is partially separated by chemical treatment or the like. The process of causing.
  • the amount of enzyme and reaction when dispersing cells so that a part of the cells remains in the cell suspension as a cell mass having the average diameter. It must be prepared in consideration of temperature, reaction time, etc., additional strength of external force, number of additions, etc. as appropriate.
  • the "suspension culture method” is one of the cell culture methods, and refers to a method in which cells are grown in a floating state in a medium.
  • the cultured cells exist as a cluster of cells aggregated in the culture medium. Therefore, when preparing a cell mass of a predetermined size, the suspension culture method is particularly preferable.
  • cell aggregation is promoted by administering a drug that inhibits or suppresses the activity of a signal transduction factor involved in the intracellular myosin signal transduction pathway into the medium, and conversely, the activity of the same factor is promoted or Cell aggregation can be suppressed by administering the amplifying agent in the medium.
  • the “cell population arrangement step” (S0102) is a step of arranging a cell population including a cell mass in the internal space of the template.
  • “arrangement” means filling the cell population including the cell mass into the inner space of the template or the cavity formed when the inner wall portion is arranged in the inner space.
  • the cell population before being placed in the template exists as a cell mass or a cell suspension in which cells are dispersed in a solution.
  • the cell clusters and cells contained in the cell population gather, and when they come into contact with each other, they are formed into a desired three-dimensional cell structure.
  • the placement method in the mold internal space is not limited.
  • the cell suspension may slowly flow into the template internal space.
  • the inflow of the cell suspension can also be carried out with the template immersed in the medium.
  • a commercially available cell suspension containing a cell mass having a predetermined size can also be used.
  • the amount of cell population filled in the mold internal space is not particularly limited. It may be appropriately determined in consideration of the mold internal space, the shape and size of the target three-dimensional cell structure, and the like.
  • the term "filling" means a state in which a cell population is placed inside a template, then allowed to stand, and the cells become close-packed by natural sedimentation. In the case of this step, for example, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 85% or more, 90% or less, 91% or less, 92% or less of the volume of the mold internal space, It may be filled up to 93% or less, 94% or less, or 95% or less.
  • the yield when producing a three-dimensional cell structure can be further improved.
  • the filling rate is less than 60% of the volume of the inner space of the mold, the gap between the contact portions between the cell masses becomes large due to the presence of the cell masses at a low density, and cracks and rough surfaces are formed in the culture step. It is not preferable because it may form a cell culture having the cells.
  • the filling rate is larger than 95% of the volume of the mold internal space, the physical load due to the contact between the cell clusters increases due to the presence of the cell clusters at an ultra-high density, which is not preferable.
  • the arrangement of the cell population in the template can be performed by one or multiple operations. In the case of a plurality of times, the second placement is performed after a predetermined time has elapsed after the first placement step.
  • the amount of cell population arranged in each round may be the same or different.
  • the cell masses and cell types that make up the cell population, or the average diameter of the cell masses may be the same or different each time.
  • the cell mass or the cell mass is arranged by a method of spontaneously dropping the cell mass or cells, a method of applying artificial pressure from the cell population inlet, or a method of suction arrangement from the outside of the template and / or the inside of the inner wall.
  • Stacking cells can be mentioned, but is not limited to these methods. However, if an external force is applied to the cells during placement, damage to the cells may occur, so placement by free fall without applying artificial pressure or suction force is preferable.
  • “stacking” means arranging a plurality of the cell masses and cells in the internal space of the template so as to maintain a state of being in contact with each other in the direction of gravity.
  • the “culturing step” is a step of culturing a cell population arranged in the template internal space after the cell population arranging step in a medium together with the template.
  • "With a template” means that a cell population arranged in a template internal space is immersed in a medium together with the template and cultured. Since the mold wall portion has a porous structure with liquid permeability, the cell population in the mold internal space can be cultured by the inflow and outflow of the medium through the pores thereof. Within the interior space, the cell masses and / or cells adhere to each other via contact and integrate within the interior space of the mold. As a result, the desired three-dimensional cell structure is formed inside the template after this step.
  • the shape and size of the culture tank containing the medium are not limited as long as the mold can be immersed in the tank.
  • the culture conditions such as the culture temperature, time, and CO 2 concentration in this step are not particularly limited. It may be done within the scope of the conventional law in the field.
  • the lower limit of the culture temperature may be 20 ° C. or higher or 35 ° C. or higher
  • the upper limit may be 45 ° C. or lower or 40 ° C. or lower, but is preferably 37 ° C.
  • the culture period may be a period during which each cell mass or cell arranged in the template internal space is integrated and a target three-dimensional cell structure is formed.
  • the CO 2 concentration at the time of culturing may be 4% or more, 4.5% or more, and 10% or less, or 5.5% or less, but is preferably 5%.
  • the medium can be changed at an appropriate frequency.
  • the frequency of medium replacement varies depending on the cell type to be cultured, the type of medium, and the volume and shape of the mold internal space, it may be appropriately determined in consideration of these conditions. For example, it may be performed once every 5 days or more, once every 4 days or more, once every 3 days or more, once every 2 days or more, and once a day or more.
  • the flow state of the medium during culture does not matter. It may be a static culture or a fluid culture, but it is preferably a fluid culture.
  • “Standing culture” means culturing the medium in a static state in a culture tank.
  • Fluid culture refers to culturing under conditions in which the medium is allowed to flow. Since the pores in the mold wall have liquid permeability, the medium can be naturally flowed in and out through the pores even in static culture, but the flow of the medium actively allows the medium to flow in and out of the mold internal space. Flow culture is preferable because it can be carried out easily. Examples of the flow culture method include a swirl culture method, a swing culture method, or a combination thereof.
  • “Swirl culture method” refers to a method of culturing under conditions in which the medium flows in the culture tank due to stress (centrifugal force, centripetal force) due to the swirling flow. Specifically, the culture tank is swirled so as to draw a closed orbit such as a circle, an ellipse, a flat circle, or a flat ellipse along a horizontal plane, or the culture tank is left to stand and stirred with a stirring rod or agitator.
  • a stirrer such as a wing can be used to swirl the medium in the tank to allow the medium to flow.
  • the swirling speed in the swirling culture method is not particularly limited, but the lower limit may be 1 rpm or more, 10 rpm or more, 50 rpm or more, 60 rpm or more, 70 rpm or more, 80 rpm or more, 85 rpm or more, or 90 rpm or more.
  • the upper limit can be 200 rpm or less, 150 rpm or less, 120 rpm or less, 115 rpm or less, 110 rpm or less, 105 rpm or less, 100 rpm or less, 95 rpm or less, or 90 rpm or less.
  • the swirling width during swirling culture is not particularly limited, but the lower limit can be, for example, 1 mm or more, 10 mm or more, 20 mm or more, or 25 mm or more.
  • the upper limit can be, for example, 200 mm or less, 100 mm or less, 50 mm or less, 30 mm or less, or 25 mm or less.
  • the "rocking culture method” refers to a method of culturing under conditions in which a rocking flow is applied to the medium by a linear reciprocating motion such as rocking stirring.
  • the culture tank is swung in a plane substantially perpendicular to the horizontal plane.
  • the rocking speed is not particularly limited, but for example, when one round trip is once, the lower limit is 2 times, 4 times, 6 times, 8 times, or 10 times per minute, while the upper limit is 15 times per minute. It may swing 20 times, 25 times, or 50 times.
  • the swing angle is not particularly limited, but for example, the lower limit is 0.1 °, 2 °, 4 °, 6 °, or 8 °, while the upper limit is 10 °, 12 °, 15 °, 18 °, or 20 °. can do. Furthermore, it is also possible to incubate while stirring by a motion that combines the above-mentioned turning and rocking.
  • the “separation step” is a step of separating the three-dimensional cell structure obtained after the culture step and the template.
  • This process is a selective process.
  • the template is composed of a material that can be fused with living tissue, for example, a polysaccharide polymer or a gelled protein
  • the three-dimensional cell structure after the culture step does not necessarily separate from the template and is in a state of being integrated with the template. It can also be used in. In this case, this step is unnecessary.
  • the template is an inanimate material such as metal or plastic, it is desirable to separate the template by this step after the culturing step and at least before using the produced three-dimensional cell structure.
  • the method for separating the three-dimensional cell structure from the template is not particularly limited. If the mold has an internal space, a three-dimensional cell structure, and an inner wall portion, a physical and / or chemical force may be applied between the inner wall portion and the three-dimensional cell structure, and both may be peeled off.
  • Physical forces include, for example, shear stress, tensile force, pressure (including water pressure and air pressure), and temperature.
  • Chemical forces include, for example, enzyme treatments such as weak proteolytic enzymes. When a physical force or a chemical external force is excessively applied to a cell, cell death can be induced, so the external force should be weak.
  • this step when this step is performed, by making the cell adhesion surface such as the inner wall surface of the mold and the surface of the inner wall a non-cell adhesion material, the target three-dimensional cell structure is formed in the inner space of the mold, but is filled. Since the cell population does not adhere to the inner space wall surface of the mold or the surface of the inner wall portion, this step can be easily achieved with almost no external force applied.
  • the cell culture taken out from the template becomes the target three-dimensional cell structure obtained by the production method of the present invention.
  • a three-dimensional cell structure having high cell density, shape uniformity and physical strength can be easily and stably produced in any shape. Can be done. As a result, the yield is improved and mass production of the three-dimensional cell structure becomes possible.
  • a second aspect of the present invention is a three-dimensional cell structure.
  • the three-dimensional cell structure of the present invention can be obtained by using the method for producing a three-dimensional cell structure of the first aspect.
  • the three-dimensional cell structure obtained by the conventional method has a low cell density, tends to have a non-uniform shape, and has insufficient strength.
  • the cells can be packed at high density by arranging the cell mass having a predetermined average diameter in the space inside the template, and then the cells are cultured together with the template. By doing so, it is possible to obtain an unprecedented three-dimensional cell structure having high cell density, shape uniformity, and physical strength.
  • the structure of the three-dimensional cell structure of the present invention has a shape homologous to the shape of the internal space in the template according to the method for producing the three-dimensional cell structure of the first aspect. Therefore, it is possible to have an arbitrary shape depending on the mold.
  • Specific examples of the shape of the three-dimensional cell structure of the present invention include a tubular structure that imitates a blood vessel described in Examples, and a structure having a complicated shape such as an auricle.
  • the cells constituting the structure are the cell population arranged in the template in the cell population arrangement step and the cells obtained by the culture step of culturing in the medium. Therefore, the type of cells constituting the structure depends on the cell mass and the type of cells contained in the cell population arranged at the time of production. In addition, individual cell masses and cells contained in a cell population are bound to each other by cell adhesion in the culture step to form an integrated cell aggregate.
  • the three-dimensional cell structure of the present invention has a significantly higher cell density than the conventional three-dimensional cell structure, and therefore has features of high shape uniformity and high physical strength.
  • the three-dimensional cell structure of the present invention can be obtained by the production method of the first aspect and can be used as a member for transplantation in regenerative medicine. That is, according to the three-dimensional cell structure of the present invention, a three-dimensional cell structure used for a member for transplantation is provided. Further, according to the three-dimensional cell structure of the present invention, the use of the three-dimensional cell structure for manufacturing a member for transplantation is provided. Further, according to the three-dimensional cell structure of the present invention, a method of transplanting a three-dimensional cell structure into a patient or subject, including a step of administering a therapeutically effective amount of the three-dimensional cell structure to the patient or subject, and a patient. Alternatively, a method of treating the subject's disease is provided.
  • transplanting member examples include International Publication WO2016 / 068292, and Anna D.A. Dikina, et al. , Biomaterials, 2015, 52: 452-462., Etc., known transplanting members suitable for artificial angioplasty and cartilage regeneration, and the three-dimensional cell structure of the present invention can also be used for such applications. it can.
  • the "patient or subject” in the present specification is typically a human, but may be another animal.
  • Other animals include, for example, rodents such as mice, rats, hamsters, guinea pigs, snails, livestock or pets such as dogs, cats, rabbits, cows, horses, pigs, sheep, goats, ferrets, and humans.
  • rodents such as mice, rats, hamsters, guinea pigs, snails, livestock or pets such as dogs, cats, rabbits, cows, horses, pigs, sheep, goats, ferrets, and humans.
  • primates such as crab monkeys, red-tailed monkeys, common marmosets, Japanese monkeys, gorillas, and chimpanzees.
  • the three-dimensional cell structure of the present invention has high cell density, shape uniformity and physical strength, it can be used as a member for transplantation in regenerative medicine.
  • fat was aseptically sucked and collected using a cannula ( ⁇ 4 / 260 mm: TP-201, ⁇ 3 / 260 mm: TP-202, MEDIKAN) connected to a disposable syringe (described above) (Lipokit, MEDICAN). .. Then, the fat was centrifuged under the condition of 700 ⁇ g for 5 minutes, and the supernatant was removed as a waste liquid.
  • a cannula ⁇ 4 / 260 mm: TP-201, ⁇ 3 / 260 mm: TP-202, MEDIKAN
  • Lipokit, MEDICAN disposable syringe
  • ⁇ MEM Alpha Modification of Minimum Essential Medium Eagle
  • FBS deactivated fetal bovine serum
  • the ratio of cells positive for the surface antigen CD90 was analyzed using a flow cytometer with respect to the cell suspension.
  • CD90 is a representative positive marker for MSCs.
  • the surface antigen analysis was carried out using FACS canto of Becton Dickinson (BD), the number of analyzed cells: 10,000 cells, and the flow velocity setting: Medium.
  • FITC Mouse IgG1, ⁇ Isotype Control (BD / model number: 550616) was used as an antibody for isotype control.
  • FITC Mouse Anti-Human CD90 (BD / model number: 555595) was used as an antibody against the CD90 antigen.
  • the cell population of the first passage was exfoliated using TrypLE Select (described above), diluted with ⁇ MEM containing 10% deactivated FBS at the final concentration, and recovered by centrifugation. ..
  • the collected cell population was suspended by adding a cryopreservation solution Bunbunker (registered trademark) (GC Lymphotech), and then cryopreserved under a -80 ° C deep freezer. Then, it was thawed and seeded in CellSTACK® (described above) at a density of 4500 cells / cm 2, and subcultured until it became subconfluent.
  • the cell population subcultured at this time is referred to as a "second passage cell population”.
  • Each surface antigen (CD90 positive rate, CD73 positive rate, CD105 positive rate, CD45 positive rate and negative rate) was measured for the cell population of the second passage using a flow cytometer. did.
  • Surface antigen analysis was performed using FACS canto (described above) with the number of cells analyzed: 10,000 cells and the flow velocity setting: Medium. In this measurement, FITC mouse IgG1, ⁇ Mouse Control (described above) or PE mouse IgG1, ⁇ Mouse Control (manufactured by BD / model number: 550617) was used as an antibody for isotype control.
  • PE anti-human CD90 (Th1) Antibody (manufactured by BioLegend / model number: 328110) was used as an antibody against the CD90 antigen
  • PE Mouse Anti-Human CD73 (manufactured by BD / model number: 550257) was used as an antibody against the CD73 antigen.
  • Anti-CD105 / FITC (manufactured by Ancell / model number: 326-040) was used as an antibody against the antigen
  • FITC Mouse Anti-Human CD45 (manufactured by BD / model number: 555482) was used as an antibody against the CD45 antigen.
  • the positive rate of CD90, CD73 and CD105 was 80% or more, and the positive rate of CD45 was less than 5% (negative rate was 95% or more). .. From the above results, it was confirmed that the cell population of the second passage was a cell population containing adipose MSC.
  • a cell mass was prepared using the cell population of the second passage.
  • ⁇ MEM containing deactivated FBS having a final concentration of 10% was used as a cell culture medium to prepare a cell suspension containing a second-generation cell population.
  • the cell suspension was seeded on a Prime Surface® plate 96U at 6.0 ⁇ 10 4 cells per well. Subsequently, the seeded plate was statically cultured at 37 ° C. for 24 hours under 5% CO 2. After culturing, the formation of cell clusters was confirmed using a microscope (manufactured by OLYMPUS).
  • the diameters of 60 cell masses are measured from any three directions based on the obtained photographed image. , Calculated.
  • the magnification of the eyepiece at the time of photographing the microscope image was 10 times, and the magnification of the objective lens was 4 times.
  • FIG. 2 shows a conceptual diagram of the prepared mold (0200).
  • the mold is a cylindrical bottom (0201) made of brass with a diameter of 5 mm and a porous mesh made of polypropylene (manufactured by Sampler Tech Co., Ltd., catalog number: WEB17815, hole major axis: 275.12 ⁇ m, average wire diameter: It has a substantially cylindrical wall portion (0202) composed of 140.74 ⁇ m and an aperture ratio of 43.8%). Further, a cylindrical inner wall portion (0203) having a diameter of 3 mm made of stainless steel is provided in the central portion of the inner space of the mold.
  • the prepared mold has a cylindrical bottom portion (0201), a substantially cylindrical wall portion (0202), and a hollow portion (0204) surrounded by a cylindrical inner wall portion (0203). The mold was fixed to a silicon base (not shown).
  • the cell suspension containing 60 cell masses obtained in (5) above is flowed into the cavity (0204) formed in the template (0200) to form the cell mass. Randomly placed in the cavity. Then, the arranged cell mass was immersed in a cell culture medium together with the template, and statically cultured at 37 ° C. for 2 weeks under 5% CO 2. As the cell culture medium, ⁇ MEM containing deactivated FBS having a final concentration of 10% was used, and the medium was exchanged every two days during the above culture period.
  • Example 1 Fat collection The basic operation followed the method described in "(1) Fat collection" of Comparative Example 1. However, in this example, fat was collected from donor B, which was different from Comparative Example 1 in which informed consent was obtained.
  • Example 2 (1) Fat collection In the basic operation, fat was collected from the same donor A as in Comparative Example 1 according to the method described in "(1) Fat collection" of Comparative Example 1. (2) Preparation of fat MSC From the enzyme treatment of the collected fat and the recovery of the fat MSC to the surface antigen analysis, the methods described in (2) to (4) of Comparative Example 1 were followed. In the surface antigen analysis, the positive rates of CD90, CD73 and CD105 in the second passage cell population were all 80% or more (specifically, CD90: 100%, CD73: 98%, CD105: 85). %). The positive rate of CD45 was less than 5% (negative rate was 95% or more) (specifically, the positive rate of CD45: 0% (negative rate: 100%)).
  • the cell population of the second passage of this example was a cell population containing adipose MSC.
  • (3) Preparation of cell mass The basic operation followed the method described in "(5) Preparation of cell mass" of Comparative Example 1. However, in this example, the cell suspension was seeded so as to have 2.0 ⁇ 10 3 cells per well of the plate after seeding the cell suspension.
  • (4) Preparation of Mold The basic operation followed the method described in "(6) Preparation of mold" of Comparative Example 1. However, in this example, the holes of the porous mesh constituting the wall portion of the mold had a major axis of 275.12 ⁇ m and an average wire diameter of 140.74 ⁇ m.
  • the aperture ratio of the porous mesh in the mold wall was calculated to be 43.8%.
  • (5) Production of three-dimensional cell structure The basic operation followed the method described in "(7) Production of three-dimensional cell structure" of Comparative Example 1. However, in this example, 138 cell masses obtained in the above-mentioned “(3) Preparation of cell mass” and 124 cell masses obtained in “(5) Preparation of cell mass” of Comparative Example 1 are used. They were mixed and randomly placed in the cavity.
  • (6) Separation of 3D cell structure from template The basic operation followed the method described in "(8) Separation of 3D cell structure from template" of Comparative Example 1.
  • ⁇ indicates the case where the crack was confirmed, and ⁇ indicates the case where the crack could not be confirmed.
  • ⁇ indicates a case where the separation was easy, and ⁇ indicates a case where the separation was difficult.
  • acquisition of the target three-dimensional cell structure indicates the case where the acquisition was possible, and ⁇ indicates the case where the acquisition was not possible.
  • homoogeneity of the three-dimensional cell structure indicates a case where it was uniform, and ⁇ indicates a case where it was non-uniform.
  • the ratio (desired diameter cell mass content) of cell clusters having a desired diameter (average diameter of 50 ⁇ m or more and 750 ⁇ m or less) in the cell population in Example 1 was 100%.
  • the desired diameter cell mass content in Example 2 was 53%.
  • the desired diameter cell mass content in Comparative Example 1 was 0%.
  • the cell population containing the cell mass could be filled up to 60% or more of the volume of the template internal space (that is, the cell mass filling rate was 60% or more).
  • the cell mass filling rate was 60% or more.
  • the cell mass could not be filled at such a high density (that is, the cell mass filling rate was less than 60%).
  • the three-dimensional cell structure is taken out from the cavity formed by the mold internal space and the inner wall portion without applying a particularly large force.
  • the three-dimensional cell structure could be grasped using tweezers, so that the three-dimensional cell structure was removed from the stainless steel cylinder. It could be easily separated.
  • Table 1 and FIG. 8 in Comparative Example 1 although an attempt was made to remove the three-dimensional cell structure from the stainless steel cylinder, a large number of cracks were found on the surface of the three-dimensional cell structure. Due to the significant lack of physical strength, it was not possible to separate the 3D cell structure from the stainless steel cylinder while maintaining its original shape.

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Abstract

The present invention addresses the problem of developing and providing a method for producing, in a simple and stable manner, a three-dimensional cell structure having an arbitrary three-dimensional shape and having high shape uniformity and physical strength.　A three-dimensional cell structure capable of solving said problem can be produced by placing a cell population that contains cell clusters having an average diameter of 50-750 µm inside a mold that has a wall surface having a liquid-permeable porous structure and by culturing the cell population therein.

Description

三次元細胞構造体の製造方法Manufacturing method of three-dimensional cell structure
　本発明は、三次元細胞構造体の製造方法に関する。 The present invention relates to a method for producing a three-dimensional cell structure.
　近年、無限に増殖できる能力と、様々な細胞に分化する能力を有した幹細胞を利用した再生医療における難治性疾患や生活習慣病等に対する根本的治療法の実用化の可能性が高まっている。例えば、インビトロ（ｉｎ ｖｉｔｒｏ）の実験系で、多能性幹細胞から神経細胞、心筋細胞、血液細胞、及び網膜細胞等、様々な細胞への分化誘導は、既に成功している（非特許文献１）。 In recent years, there is an increasing possibility of practical application of a fundamental treatment method for intractable diseases and lifestyle-related diseases in regenerative medicine using stem cells having the ability to proliferate infinitely and differentiate into various cells. For example, in an in vitro experimental system, induction of differentiation from pluripotent stem cells into various cells such as nerve cells, myocardial cells, blood cells, and retinal cells has already been successful (Non-Patent Document 1). ).
　一方で、幹細胞を用いた各種臓器や器官の再生に関しては、実用化に向けてまだ多くの課題が残されている。その一つが幹細胞由来の立体構造物の構築における問題である。再生医療技術によって構築された人工臓器や人工器官を、機能不全や欠損等によりその機能が失われた臓器や器官に代わる移植用部材として用いるには、十分な強度と代替する臓器や器官を模した複雑な立体形状を有する必要がある。 On the other hand, there are still many issues to be solved for practical use regarding the regeneration of various organs and organs using stem cells. One of them is the problem in the construction of stem cell-derived three-dimensional structures. To use an artificial organ or artificial organ constructed by regenerative medicine technology as a transplant member to replace an organ or organ whose function has been lost due to malfunction or defect, etc., it imitates an organ or organ that has sufficient strength and substitutes. It is necessary to have a complicated three-dimensional shape.
　この問題を解決するために、培養幹細胞を所望の形状に成形する様々な方法が開発されている。 In order to solve this problem, various methods for shaping cultured stem cells into a desired shape have been developed.
　例えば、特許文献１では、細胞粒子を規制枠部に投入し、平板状の組織体を形成させた後、押圧成形する方法が開示されている。外圧という外部刺激を負荷することで、強度及び生体適合性の高い細胞構造体を形成することができる。また、押圧成形部に溝部、貫通孔、屈曲状に湾曲した形状等を設けることで、所望の形状を有する細胞構造体を得ることができる。 For example, Patent Document 1 discloses a method in which cell particles are put into a regulation frame portion to form a flat tissue and then press-molded. By applying an external stimulus called external pressure, a cell structure with high strength and biocompatibility can be formed. Further, a cell structure having a desired shape can be obtained by providing a groove portion, a through hole, a curved shape in a bent shape, or the like in the press molding portion.
　特許文献２では、高分子入りの細胞構造体（モザイク細胞塊）を原料に、シリコン製の鋳型を用いて管状構造体を成形する方法が開示されている。下部土台の芯受けに液抜けの穴を４箇所程開けて液拡散性を高め、細胞の生存を高める工夫がみられる。 Patent Document 2 discloses a method of molding a tubular structure using a silicon template using a cell structure containing a polymer (mosaic cell mass) as a raw material. There are four holes in the core holder of the lower base to improve the liquid diffusivity and improve the survival of cells.
　特許文献３では、細胞を管状に成形するために、リング状に配置した剣山に細胞塊を貫通させて固定成形した後に培養を行うことで目的の形状を有する細胞構造体を得る方法が開示されている。 Patent Document 3 discloses a method for obtaining a cell structure having a desired shape by culturing a cell mass after penetrating a cell mass through a ring-shaped sword ridge in order to form the cell into a tubular shape. ing.
　非特許文献２では、シングルセルを原料として、アガロースゲル製の鋳型で成形する方法が開示されている。 Non-Patent Document 2 discloses a method of molding a single cell as a raw material using a mold made of agarose gel.
特開２０１６－００００２９JP 2016-0000029 ＷＯ２０１６／０６８２９２WO2016 / 068292 ＷＯ２０１３／０７３６７２WO2013 / 073672
　上記先行技術文献に開示された三次元細胞構造体の製造方法には、以下の課題が存在する。 The method for producing a three-dimensional cell structure disclosed in the above prior art document has the following problems.
　特許文献１に記載の方法で使用される鋳型は、押圧成形をするため平板状であり、培養液の拡散性を高める機構を備えていない。そのため、任意の形状の組織体を容易に製造することはできないという問題がある。また、培養液の拡散性が低いため組織内部への栄養供給が不十分になりやすいという問題もある。さらに、押圧成形で細胞に外部刺激を加えることが組織体の機能に悪影響を及ぼすリスクを伴う。 The mold used by the method described in Patent Document 1 is flat because it is pressed and molded, and does not have a mechanism for increasing the diffusivity of the culture solution. Therefore, there is a problem that a structure having an arbitrary shape cannot be easily manufactured. In addition, there is also a problem that the nutrient supply to the inside of the tissue tends to be insufficient because the diffusivity of the culture solution is low. In addition, applying external stimuli to cells by press molding carries the risk of adversely affecting tissue function.
　特許文献２に記載の方法で使用される鋳型は筒状構造を有するため、三次元細胞構造体として管状構造物を製造することはできる。しかし、任意の形状の構造体を製造することはできないという問題がある。また、鋳型に設けられた液抜けの孔だけでは管状三次元細胞構造体内部への栄養供給が依然として十分ではないという問題もある。 Since the template used by the method described in Patent Document 2 has a tubular structure, a tubular structure can be produced as a three-dimensional cell structure. However, there is a problem that a structure having an arbitrary shape cannot be manufactured. In addition, there is also a problem that the nutrient supply to the inside of the tubular three-dimensional cell structure is still insufficient only by the drainage holes provided in the mold.
　特許文献３に記載の方法は、管状剣山に細胞塊を個々に固定する必要があるため生産性が著しく低いという問題がある。また、三次元細胞構造体の形状が簡易なものに限定される上に、得られる三次元細胞構造体の細胞密度が低く、形状が不均一となりやすいという問題もある。さらに、細胞塊を剣山で突き刺し固定するため、細胞への物理的負荷が大きく、細胞死を誘導しやすいという問題もある。 The method described in Patent Document 3 has a problem that the productivity is extremely low because it is necessary to individually fix the cell mass to the tubular sword ridge. Further, the shape of the three-dimensional cell structure is limited to a simple one, and there is also a problem that the cell density of the obtained three-dimensional cell structure is low and the shape tends to be non-uniform. Furthermore, since the cell mass is pierced and fixed with a sword mountain, there is a problem that the physical load on the cell is large and cell death is easily induced.
　非特許文献２に記載の方法は、シングルセルを原料とするため、得られる三次元細胞構造体は低細胞密度で形状が不均一となりやすく、細胞構造体の性能や歩留りに問題がある。 Since the method described in Non-Patent Document 2 uses a single cell as a raw material, the obtained three-dimensional cell structure tends to have a low cell density and a non-uniform shape, and there is a problem in the performance and yield of the cell structure.
　上記課題を解決するために本発明者らが鋭意研究を行った結果、特定の範囲に平均直径を有する細胞塊を、通液性のある多孔構造の壁面を有した鋳型内部に配置し、培養することで、任意の三次元形状を有し、形状均一性と物理的強度が高い細胞構造体を簡便、かつ安定的に製造することに成功した。本発明は、これらの研究結果に基づいて完成に至ったものであり、以下を提供する。 As a result of diligent research conducted by the present inventors to solve the above problems, a cell mass having an average diameter in a specific range is placed inside a mold having a wall surface having a porous structure with liquid permeability and cultured. By doing so, we succeeded in easily and stably producing a cell structure having an arbitrary three-dimensional shape and having high shape uniformity and physical strength. The present invention has been completed based on these research results, and the following are provided.
（１）三次元細胞構造体の製造方法であって、平均直径が５０μｍ以上、７５０μｍ以下の細胞塊を含む細胞集団を、鋳型の内部空間内に配置する細胞集団配置工程、及び前記細胞集団を前記鋳型と共に培地中で培養する培養工程を含み、前記鋳型の壁部が通液可能な多孔構造を有する、前記製造方法。
（２）前記細胞集団が前記細胞塊を５０％以上含む、（１）に記載の製造方法。
（３）前記鋳型の内部空間が円筒状である、（１）又は（２）に記載の製造方法。
（４）前記鋳型が、鋳型内部に内壁部を有する、（１）～（３）のいずれか一に記載の製造方法。
（５）前記鋳型の内部空間と内壁部によって形成される空洞部がドーナツ状又は管状である、（４）に記載の製造方法。
（６）前記細胞塊の平均直径（μｍ）に対する前記多孔構造における孔の長径（μｍ）及び／又は最大対角線（μｍ）との比率（細胞塊の平均直径／孔の長径及び／又は最大対角線）が１．０より大きく、３．０以下である、（１）～（５）のいずれか一に記載の製造方法。
（７）前記多孔構造における開孔率が４０％以上、９０％以下である、（１）～（６）のいずれか一に記載の製造方法。
（８）前記細胞集団配置工程において、前記細胞塊を含む細胞集団を前記鋳型内部空間の体積の６０％以上、９５％以下まで充填する、（１）～（７）のいずれか一に記載の製造方法。
（９）前記細胞集団配置工程において、前記鋳型に人為的圧力を付与することなく前記細胞塊を積層する、（１）～（８）のいずれか一に記載の製造方法。
（１０）前記培養工程における培養期間が４時間以上、２８日未満である、（１）～（９）のいずれか一に記載の製造方法。
（１１）前記培養工程後に得られる三次元細胞構造体と前記鋳型とを分離する分離工程をさらに含む、（１）～（１０）のいずれか一に記載の製造方法。
（１２）前記鋳型の細胞接触面が細胞非接着素材で構成されている、（１１）に記載の製造方法。
（１３）前記細胞集団配置工程に先立ち、平均直径が５０μｍ以上、７５０μｍ以下の細胞塊を含む細胞集団を調製する細胞集団調製工程をさらに含む、（１）～（１２）のいずれか一に記載の製造方法。
（１４）前記細胞塊を構成する細胞が、間葉系幹細胞、線維芽細胞、内皮細胞及び軟骨細胞からなる群より少なくとも一つ選択される、（１）～（１３）のいずれか一に記載の製造方法。
（１５）前記間葉系幹細胞が、骨髄、脂肪、胎児付属物又は歯髄に由来するものである、（１４）に記載の製造方法。
（１６）三次元細胞構造体であって、平均直径が５０μｍ以上、７５０μｍ以下の細胞塊を含む細胞集団を、鋳型の内部空間内に配置した後、前記細胞集団を前記鋳型と共に培地中で培養して得られ、前記鋳型の壁部が通液可能な多孔構造を有する、前記三次元細胞構造体。
　本明細書は本願の優先権の基礎となる日本国特許出願番号2019-178749号の開示内容を包含する。 (1) A method for producing a three-dimensional cell structure, wherein a cell population arranging step of arranging a cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less in the internal space of a template, and the cell population are described. The production method comprising a culturing step of culturing in a medium together with the mold, wherein the wall portion of the mold has a porous structure through which liquid can pass.
(2) The production method according to (1), wherein the cell population contains 50% or more of the cell mass.
(3) The production method according to (1) or (2), wherein the internal space of the mold is cylindrical.
(4) The production method according to any one of (1) to (3), wherein the mold has an inner wall portion inside the mold.
(5) The manufacturing method according to (4), wherein the hollow portion formed by the internal space and the inner wall portion of the mold is donut-shaped or tubular.
(6) Ratio of pore major axis (μm) and / or maximum diagonal line (μm) in the porous structure to the average diameter (μm) of the cell mass (average cell mass diameter / pore major axis and / or maximum diagonal line) The production method according to any one of (1) to (5), wherein is greater than 1.0 and less than or equal to 3.0.
(7) The production method according to any one of (1) to (6), wherein the pore opening ratio in the porous structure is 40% or more and 90% or less.
(8) The method according to any one of (1) to (7), wherein in the cell population placement step, the cell population containing the cell mass is filled to 60% or more and 95% or less of the volume of the mold internal space. Production method.
(9) The production method according to any one of (1) to (8), wherein in the cell population placement step, the cell mass is laminated without applying artificial pressure to the template.
(10) The production method according to any one of (1) to (9), wherein the culture period in the culture step is 4 hours or more and less than 28 days.
(11) The production method according to any one of (1) to (10), further comprising a separation step of separating the three-dimensional cell structure obtained after the culture step and the template.
(12) The production method according to (11), wherein the cell contact surface of the template is made of a cell non-adhesive material.
(13) The method according to any one of (1) to (12), further comprising a cell population preparation step of preparing a cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less prior to the cell population placement step. Manufacturing method.
(14) The cell according to any one of (1) to (13), wherein at least one cell constituting the cell mass is selected from the group consisting of mesenchymal stem cells, fibroblasts, endothelial cells and chondrocytes. Manufacturing method.
(15) The production method according to (14), wherein the mesenchymal stem cells are derived from bone marrow, fat, fetal appendages or dental pulp.
(16) A cell population which is a three-dimensional cell structure and contains a cell mass having an average diameter of 50 μm or more and 750 μm or less is placed in the inner space of a template, and then the cell population is cultured in a medium together with the template. The three-dimensional cell structure obtained by the above method, wherein the wall portion of the template has a porous structure through which liquid can pass.
This specification includes the disclosure of Japanese Patent Application No. 2019-178749, which is the basis of the priority of the present application.
　本発明の三次元細胞構造体の製造方法によれば、任意の三次元形状を有する均一性の高い細胞構造体を簡便に製造することができる。
　本発明の三次元細胞構造体の製造方法によれば、形状均一性と物理的強度の高い三次元細胞構造体を安定的に製造することができる。 According to the method for producing a three-dimensional cell structure of the present invention, a highly uniform cell structure having an arbitrary three-dimensional shape can be easily produced.
According to the method for producing a three-dimensional cell structure of the present invention, a three-dimensional cell structure having high shape uniformity and high physical strength can be stably produced.
本発明の三次元細胞構造体の製造フローを示す図である。It is a figure which shows the manufacturing flow of the 3D cell structure of this invention. 本発明の三次元細胞構造体製造方法で使用する鋳型の一例を示す図である。It is a figure which shows an example of the template used in the 3D cell structure manufacturing method of this invention. 実施例１の細胞集団配置工程後の培養工程における細胞培養物の様子を観察した結果を示した図である。Ａは細胞集団配置工程直後の図、またＢは培養７日目の図を示す。図中、矢印は配置した細胞集団（主に細胞塊）を示す。It is a figure which showed the result of observing the state of the cell culture in the culture step after the cell population arrangement step of Example 1. FIG. A is a diagram immediately after the cell population placement step, and B is a diagram on the 7th day of culturing. In the figure, the arrows indicate the arranged cell population (mainly the cell mass). 実施例２の細胞集団配置工程後の培養工程における細胞培養物の様子を観察した結果を示した図である。Ａは細胞集団配置工程直後の図、またＢは培養７日目の図を示す。図中、矢印は配置した細胞集団（主に細胞塊）を示す。It is a figure which showed the result of observing the state of the cell culture in the culture step after the cell population arrangement step of Example 2. FIG. A is a diagram immediately after the cell population placement step, and B is a diagram on the 7th day of culturing. In the figure, the arrows indicate the arranged cell population (mainly the cell mass). 比較例１の細胞集団配置工程後の培養工程における細胞培養物の様子を観察した結果を示した図である。Ａは細胞集団配置工程直後の図、またＢは培養７日目の図を示す。図中、矢印は配置した細胞集団（主に細胞塊）を示す。It is a figure which showed the result of observing the state of the cell culture in the culture step after the cell population arrangement step of the comparative example 1. FIG. A is a diagram immediately after the cell population placement step, and B is a diagram on the 7th day of culturing. In the figure, the arrows indicate the arranged cell population (mainly the cell mass). 実施例２の鋳型抜去時の組織体（矢印）の様子を確認した結果を示した図である。図中、矢頭は鋳型内部空間に内壁部として設置したステンレス製円柱を示す。It is a figure which showed the result of having confirmed the state of the structure (arrow) at the time of removing the mold of Example 2. In the figure, the arrowhead indicates a stainless steel cylinder installed as an inner wall in the space inside the mold. 実施例２の鋳型及び内壁部抜去後の三次元細胞構造体の状態を示した図である。It is a figure which showed the state of the three-dimensional cell structure after removal of the template and the inner wall part of Example 2. FIG. 比較例１の鋳型抜去時の組織体（矢印）の状態を示した図である。図中、矢頭は、鋳型内部空間に内壁部として設置したステンレス製円柱である。It is a figure which showed the state of the tissue (arrow) at the time of removing the mold of the comparative example 1. FIG. In the figure, the arrowhead is a stainless steel cylinder installed as an inner wall in the space inside the mold.
１．三次元細胞構造体の製造方法
１－１．概要
　本発明の第１の態様は、三次元細胞構造体の製造方法である。本発明の製造方法では、平均直径が特定の範囲にある細胞塊を包含する細胞集団を、通液性のある多孔構造の壁面を有する鋳型内部に配置する。それにより、内部空間内に細胞塊を高密度で配置することが可能となる。また、鋳型内部空間に細胞集団を充填、積層するのみで足り、それ故に細胞に対する外圧等の物理的負荷がないという利点がある。本発明の製造方法によれば、任意の三次元形状を有する細胞密度の高い細胞構造体を簡便に製造することができる。それにより、強度と均一性が高い三次元細胞構造体を安定的に製造することが可能となり、歩留りが向上し、三次元細胞構造体の大量生産が可能となる。 1. 1. Method for producing three-dimensional cell structure 1-1. Outline The first aspect of the present invention is a method for producing a three-dimensional cell structure. In the production method of the present invention, a cell population containing a cell mass having an average diameter in a specific range is placed inside a mold having a wall surface having a porous structure with fluid permeability. As a result, cell clusters can be arranged at high density in the internal space. Further, it is sufficient to fill and stack the cell population in the space inside the template, and therefore there is an advantage that there is no physical load such as external pressure on the cells. According to the production method of the present invention, a cell structure having a high cell density having an arbitrary three-dimensional shape can be easily produced. As a result, it becomes possible to stably produce a three-dimensional cell structure having high strength and uniformity, improve the yield, and enable mass production of the three-dimensional cell structure.
１－２．用語の定義及びその構成
　本明細書で頻用する以下の用語について定義し、その構成について具体的に説明をする。なお、特に断りのない限り、本項に記載の以下の定義は、本発明の他の態様においても共通する。 1-2. Definition of terms and their structure The following terms frequently used in the present specification are defined, and their structures are specifically described. Unless otherwise specified, the following definitions described in this section are common to other aspects of the present invention.
（１）三次元細胞構造体
　本明細書において「三次元細胞構造体」とは、細胞集団によって構成される立体構造物をいう。したがって、細胞シートのような細胞層からなり、その厚さがほぼ均一な平面構造物は含まない。三次元細胞構造体は、細胞のみで構成されていてもよいが、細胞以外の物質を包含していてもよい。細胞以外の物質としては、例えば、細胞外基質、鋳型の一部等をあげることができるが、これらに限定されない。また、三次元細胞構造体は、スキャフォールドを有していてもよいし、スキャフォールドフリーでもよい。好ましくはスキャフォールドフリーである。本明細書における「スキャフォールド」は、細胞を増殖させるための足場を意味する。スキャフォールドは、生体に吸収される素材から構成されることが好ましく、そのような素材としては、例えば、ポリ乳酸、コラーゲン等が挙げられるが、これらに限定されない。三次元細胞構造体を構成する各細胞は、同一の、又は異なる種類でのいずれであってもよい。 (1) Three-dimensional cell structure As used herein, the term "three-dimensional cell structure" refers to a three-dimensional structure composed of a cell population. Therefore, it is composed of a cell layer such as a cell sheet and does not include a planar structure having a substantially uniform thickness. The three-dimensional cell structure may be composed only of cells, but may contain substances other than cells. Examples of substances other than cells include, but are not limited to, extracellular matrix, a part of a template, and the like. Further, the three-dimensional cell structure may have a scaffold or may be scaffold-free. It is preferably scaffold-free. As used herein, "scaffold" means a scaffold for growing cells. The scaffold is preferably composed of a material that is absorbed by a living body, and examples of such a material include, but are not limited to, polylactic acid, collagen, and the like. Each cell constituting the three-dimensional cell structure may be of the same or different types.
　三次元細胞構造体の形状は、定型形状、又は不定形状のいずれでもよい。定型形状としては、例えば、球形、楕円体形、立方形、直方形、多面体形、円錐形、角錐形、円柱形、角柱形、螺旋形、ドーナッツ状形状又は管状形状を挙げることができるが、これらに限定されない。 The shape of the three-dimensional cell structure may be either a fixed shape or an indefinite shape. Examples of the standard shape include a spherical shape, an elliptical shape, a cubic shape, a rectangular shape, a polyhedral shape, a conical shape, a pyramidal shape, a cylindrical shape, a prismatic shape, a spiral shape, a donut shape shape, and a tubular shape. Not limited to.
（２）細胞
　本明細書における「細胞」は、三次元細胞構造体の構成単位であり、特に断りの無い限り、細胞間接着、又は細胞－細胞外基質間接着が可能な接着性細胞をいう。接着性細胞が前記細胞培養基材、細胞、又は細胞外基質と接着することを「細胞接着」という。 (2) Cell The “cell” in the present specification is a constituent unit of a three-dimensional cell structure, and means an adhesive cell capable of cell-cell adhesion or cell-extracellular matrix adhesion unless otherwise specified. .. Adhesion of adherent cells to the cell culture substrate, cells, or extracellular matrix is referred to as "cell adhesion".
　本明細書における細胞の種類は限定しない。例えば、生体組織を構成する細胞、生体組織を構成する細胞から派生した細胞、幹細胞、又は幹細胞から分化した細胞が挙げられる。また、本明細書における細胞は、生体組織から単離された細胞であってもよい。 The type of cell in the present specification is not limited. For example, cells constituting a living tissue, cells derived from cells constituting a living tissue, stem cells, or cells differentiated from stem cells can be mentioned. Moreover, the cell in this specification may be a cell isolated from a living tissue.
　本明細書において「生体組織」とは、生物の生体を構成する各種組織をいう。例えば、上皮組織、内皮組織、結合組織、筋組織、及び神経組織等が挙げられる。それらの組織を構成する細胞として、例えば、上皮細胞、内皮細胞、線維芽細胞、筋細胞、神経細胞、及び軟骨細胞が挙げられる。 In the present specification, the "living tissue" refers to various tissues constituting the living body of an organism. For example, epithelial tissue, endothelial tissue, connective tissue, muscle tissue, nerve tissue and the like can be mentioned. Examples of cells constituting these tissues include epithelial cells, endothelial cells, fibroblasts, muscle cells, nerve cells, and chondrocytes.
　「幹細胞（ｓｔｅｍ ｃｅｌｌ）」とは、様々な細胞への分化能、及び自己複製能を持つ細胞をいう。例えば、成体幹細胞、及び多能性幹細胞等が挙げられる。 "Stem cell" refers to a cell that has the ability to differentiate into various cells and the ability to self-renew. For example, adult stem cells, pluripotent stem cells and the like can be mentioned.
　「成体幹細胞（ａｄｕｌｔ ｓｔｅｍ ｃｅｌｌ）」とは、成体の各組織中に存在し、最終分化が未完了で、ある程度の多分化能を有する幹細胞であって、体性幹細胞（ｓｏｍａｔｉｃ ｓｔｅｍ ｃｅｌｌ）又は組織性幹細胞（ｔｉｓｓｕｅ ｓｔｅｍ ｃｅｌｌ）とも呼ばれる。例えば、神経幹細胞、腸管上皮幹細胞、造血幹細胞、毛包幹細胞、色素幹細胞、間葉系幹細胞等が挙げられる。「神経幹細胞」とは、主に神経細胞、及びグリア細胞への分化能を有する細胞である。「腸管上皮幹細胞」とは、主に小腸や大腸等の消化管内壁を構成する上皮細胞への分化能を有する細胞である。「造血幹細胞」とは、主に赤血球、白血球、血小板等の血液細胞への分化能を有する細胞である。 An "adult stem cell" is a stem cell that exists in each adult tissue, has not completed final differentiation, and has a certain degree of pluripotency, and is a somatic stem cell or tissue. It is also called a sex stem cell. Examples thereof include neural stem cells, intestinal epithelial stem cells, hematopoietic stem cells, hair follicle stem cells, pigment stem cells, mesenchymal stem cells and the like. "Neural stem cells" are mainly nerve cells and cells having the ability to differentiate into glial cells. "Intestinal epithelial stem cells" are cells having the ability to differentiate into epithelial cells that mainly constitute the inner wall of the digestive tract such as the small intestine and the large intestine. "Hematopoietic stem cells" are cells having the ability to differentiate into blood cells such as erythrocytes, leukocytes, and platelets.
　「毛包幹細胞」とは、毛幹細胞及び毛根鞘細胞等の毛包上皮性細胞の他、脂腺細胞、基底細胞への分化能を有する細胞である。また、「色素幹細胞」とは、主に色素細胞への分化能を有する細胞である。本明細書で使用する成体幹細胞は、市販の細胞又は分譲を受けた細胞を用いてもよいし、新たに作製した細胞を用いてもよい。 "Hair follicle stem cells" are cells having the ability to differentiate into hair follicle epithelial cells such as hair follicle stem cells and hair root sheath cells, as well as fat gland cells and basal cells. Further, the "chromophore stem cell" is a cell having an ability to differentiate into a chromatophore mainly. As the adult stem cells used in the present specification, commercially available cells or cells that have been distributed may be used, or newly prepared cells may be used.
　「間葉系幹細胞（Ｍｅｓｅｎｃｈｙｍａｌ Ｓｔｅｍ Ｃｅｌｌｓ；ＭＳＣｓ）」とは、標準培地での培養条件でプラスチックに接着性を示し、また表面抗原であるＣＤ７３（５－Ｎｕｃｌｅｏｔｉｄａｓｅ）及びＣＤ９０（Ｔｈｙ－１）が陽性を呈し、ＣＤ４５（ＰＴＰＲＣ（Ｐｒｏｔｅｉｎ　ｔｙｒｏｓｉｎｅ　ｐｈｏｓｐｈａｔａｓｅ，ｒｅｃｅｐｔｏｒ　ｔｙｐｅ，Ｃ））が陰性を呈する細胞である。間葉系幹細胞は、骨芽細胞、脂肪細胞、筋細胞、軟骨細胞等の間葉系に属する細胞への分化能を有してもよい。間葉系幹細胞は、例えば、骨髄、脂肪、胎児付属物、又は歯髄等から採取することができる。「胎児付属物」とは、胎児が子宮内で発育するために必要な器官であり、胎盤、臍帯、羊膜、卵膜、絨毛膜、脱落膜、羊水等が挙げられる。本明細書で使用する間葉系幹細胞は、遺伝子組み換えされた又は遺伝子組み換えされていない間葉系幹細胞であり、好ましくは、遺伝子組み換えされていない間葉系幹細胞である。 "Mesenchymal stem cells (MSCs)" show adhesion to plastic under culture conditions in standard medium, and are positive for surface antigens CD73 (5-Nucleotidase) and CD90 (Thy-1). , And CD45 (PTPRC (Protein tyrosine phosphatase, receptor type, C)) is a negative cell. Mesenchymal stem cells may have the ability to differentiate into cells belonging to the mesenchymal system such as osteoblasts, adipocytes, muscle cells, and chondrocytes. Mesenchymal stem cells can be collected from, for example, bone marrow, fat, fetal appendages, pulp and the like. The "fetal appendage" is an organ necessary for the foetation to develop in the uterus, and examples thereof include placenta, umbilical cord, amniotic membrane, egg membrane, chorion, decidua, and amniotic fluid. The mesenchymal stem cells used herein are mesenchymal stem cells that have been genetically modified or have not been genetically modified, and are preferably non-genetically modified mesenchymal stem cells.
　本明細書における「表面抗原が陽性を呈する細胞の比率」とは、フローサイトメトリーによって解析した表面抗原について陽性である細胞の比率を示す。本明細書において、「表面抗原が陽性を呈する細胞の比率」は「陽性率」と記載されることがある。また、本明細書における「表面抗原が陰性を呈する細胞の比率」とは、フローサイトメトリーによって解析した表面抗原について陰性である細胞の比率を示す。本明細書において、「表面抗原が陰性を呈する細胞の比率」は「陰性率」と記載されることがある。なお、各表面抗原に対して陰性である細胞の比率（陰性率）は、「陰性率（％）＝１００－陽性率」の式により算出することができる。 The "ratio of cells positive for surface antigen" in the present specification indicates the ratio of cells positive for surface antigen analyzed by flow cytometry. In the present specification, "the ratio of cells in which the surface antigen is positive" may be described as "positive rate". Further, the “ratio of cells in which the surface antigen is negative” in the present specification indicates the ratio of cells in which the surface antigen is negative in the surface antigen analyzed by flow cytometry. In the present specification, "the ratio of cells in which the surface antigen is negative" may be described as "negative rate". The ratio of cells negative to each surface antigen (negative rate) can be calculated by the formula "negative rate (%) = 100-positive rate".
　「多能性幹細胞」とは、生体を構成する全ての種類の細胞に分化することができる多分化能（多能性）を有し、適切な条件下のインビトロ（ｉｎ ｖｉｔｒｏ）での培養において多能性を維持したまま無限に増殖を続けることができる細胞をいう。例えば、胚性幹細胞（ＥＳ細胞：ｅｍｂｒｙｏｎｉｃ ｓｔｅｍ ｃｅｌｌ）、胚性生殖幹細胞（ＥＧ細胞：ｅｍｂｒｙｏｎｉｃ ｇｅｒｍ ｃｅｌｌ）、生殖系幹細胞（ＧＳ細胞：Ｇｅｒｍｌｉｎｅ ｓｔｅｍ ｃｅｌｌ)、そして人工多能性幹細胞（ｉＰＳ細胞：ｉｎｄｕｃｅｄ ｐｌｕｒｉｐｏｔｅｎｔ ｓｔｅｍ ｃｅｌｌｓ）等が挙げられる。「ＥＳ細胞」とは、初期胚より調製された多能性幹細胞である。「ＥＧ細胞」とは、胎児の始原生殖細胞より調製された多能性幹細胞である（Ｓｈａｍｂｌｏｔｔ Ｍ．Ｊ．ｅｔ ａｌ．，１９９８，Ｐｒｏｃ． Ｎａｔｌ. Ａｃａｄ．Ｓｃｉ．ＵＳＡ．，９５：１３７２６－１３７３１）。「ＧＳ細胞」とは、細胞精巣より調製された多能性幹細胞である（Ｃｏｎｒａｄ Ｓ．， ２００８，Ｎａｔｕｒｅ, ４５６：３４４－３４９）。また、「ｉＰＳ細胞」とは、分化済みの体細胞に少数の初期化因子をコードする遺伝子を導入することによって体細胞を未分化状態にするリプログラミングが可能となった多能性幹細胞をいう。 A "pluripotent stem cell" is a pluripotent (pluripotent) cell capable of differentiating into all types of cells constituting a living body, and is cultured in vitro under appropriate conditions. A cell that can continue to grow indefinitely while maintaining pluripotency. For example, embryonic stem cells (ES cells: embryonic stem cells), embryonic germ stem cells (EG cells: embryonic stem cells), germline stem cells (GS cells: Germline stem cells), and induced pluripotent stem cells (iPS cells: induced). Pluripotent stem cells) and the like. "ES cells" are pluripotent stem cells prepared from early embryos. "EG cells" are pluripotent stem cells prepared from fetal primordial germ cells (Shamblott MJ et al., 1998, Proc. Natl. Acad. Sci. USA., 95: 13726-13731. ). "GS cells" are pluripotent stem cells prepared from the cell testis (Conrad S., 2008, Nature, 456: 344-349). The "iPS cell" refers to a pluripotent stem cell that can be reprogrammed to bring the somatic cell into an undifferentiated state by introducing a gene encoding a small number of reprogramming factors into the differentiated somatic cell. ..
　本明細書で使用する多能性幹細胞は、市販の細胞又は分譲を受けた細胞を用いてもよいし、新たに作製した細胞を用いてもよい。なお、限定はしないが、本明細書の各発明に用いる場合、多能性幹細胞は、ｉＰＳ細胞又はＥＳ細胞が好ましい。 As the pluripotent stem cell used in the present specification, a commercially available cell or a cell for sale may be used, or a newly prepared cell may be used. Although not limited, iPS cells or ES cells are preferable as the pluripotent stem cells when used in each invention of the present specification.
　本明細書で使用するｉＰＳ細胞が市販品の場合、限定はしないが、例えば２５３Ｇ１株、２０１Ｂ６株、２０１Ｂ７株、４０９Ｂ２株、４５４Ｅ２株、ＨｉＰＳ－ＲＩＫＥＮ－１Ａ株、ＨｉＰＳ－ＲＩＫＥＮ－２Ａ株、ＨｉＰＳ－ＲＩＫＥＮ－１２Ａ株、Ｎｉｐｓ－Ｂ２株、ＴｋＤＮ４－Ｍ株、ＴｋＤＡ３－１株、ＴｋＤＡ３－２株、ＴｋＤＡ３－４株、ＴｋＤＡ３－５株、ＴｋＤＡ３－９株、ＴｋＤＡ３－２０株、ｈｉＰＳＣ ３８－２株、ＭＳＣ－ｉＰＳＣ１株、ＢＪ－ｉＰＳＣ１株等を使用することができる。また、新たに作製された臨床グレードのｉＰＳ細胞を用いてもよい。 When the iPS cells used in the present specification are commercially available products, for example, 253G1 strain, 201B6 strain, 201B7 strain, 409B2 strain, 454E2 strain, HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS -RIKEN-12A strain, Nippons-B2 strain, TkDN4-M strain, TkDA3-1 strain, TkDA3-2 strain, TkDA3-4 strain, TkDA3-5 strain, TkDA3-9 strain, TkDA3-20 strain, hiPSC38-2 A strain, MSC-iPSC1 strain, BJ-iPSC1 strain and the like can be used. In addition, newly prepared clinical grade iPS cells may be used.
　また、本明細書で使用するｉＰＳ細胞が新たに調製された細胞の場合、導入される初期化因子の遺伝子の組み合わせは、限定はしないが、例えばＯＣＴ３／４遺伝子、ＫＬＦ４遺伝子、ＳＯＸ２遺伝子及びｃ－Ｍｙｃ遺伝子の組み合わせ（Ｙｕ Ｊ，ｅｔ ａｌ．２００７，Ｓｃｉｅｎｃｅ, ３１８：１９１７－２０．）、ＯＣＴ３／４遺伝子、ＳＯＸ２遺伝子、ＬＩＮ２８遺伝子及びＮａｎｏｇ遺伝子の組み合わせ（Ｔａｋａｈａｓｈｉ Ｋ，ｅｔ ａｌ. ２００７，Ｃｅｌｌ，１３１：８６１‐７２．）を使用することができる。これらの因子の細胞への導入形態は特に限定されないが、例えば、プラスミドを用いた遺伝子導入、合成ＲＮＡの導入、タンパク質としての直接導入等が挙げられる。また、ｍｉｃｒｏＲＮＡやＲＮＡ、低分子化合物等を用いた方法で作製されたｉＰＳ細胞を用いてもよい。さらに、新たに作製された臨床グレードのｉＰＳ細胞を用いてもよい。 When the iPS cells used in the present specification are newly prepared cells, the combination of genes of the reprogramming factors to be introduced is not limited, but is, for example, OCT3 / 4 gene, KLF4 gene, SOX2 gene and c. -Myc gene combination (YuJ, et al. 2007, Science, 318: 1917-20.), OCT3 / 4 gene, SOX2 gene, LIN28 gene and Nanog gene combination (Takahashi K, et al. 2007, Cell, 131: 861-72.) Can be used. The form of introduction of these factors into cells is not particularly limited, and examples thereof include gene transfer using a plasmid, introduction of synthetic RNA, and direct introduction as a protein. Further, iPS cells prepared by a method using microRNA, RNA, low molecular weight compounds and the like may be used. In addition, newly prepared clinical grade iPS cells may be used.
　本明細書で使用するＥＳ細胞が市販品の場合、限定はしないが、例えばＫｈＥＳ－１株、ＫｈＥＳ－２株、ＫｈＥＳ－３株、ＫｈＥＳ－４株、ＫｈＥＳ－５株、ＳＥＥＳ１株、ＳＥＥＳ２株、ＳＥＥＳ３株、ＳＥＥＳ－４株、ＳＥＥＳ－５株、ＳＥＥＳ－６株、ＳＥＥＳ－７株、ＨＵＥＳ８株、ＣｙＴ４９株、Ｈ１株、Ｈ９株、ＨＳ－１８１株等を使用することができる。 When the ES cells used in the present specification are commercially available products, for example, KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SEES1 strain, and SEES2 strain are not limited. , SEES3 strain, SES-4 strain, SES-5 strain, SES-6 strain, SES-7 strain, HUES8 strain, CyT49 strain, H1 strain, H9 strain, HS-181 strain and the like can be used.
　本明細書における細胞の由来生物種は、多細胞生物であればよい。好ましくは動物、より好ましくは哺乳動物である。例えば、マウス、ラット、ハムスター、モルモット、スナネズミ等のげっ歯類、イヌ、ネコ、ウサギ、ウシ、ウマ、ブタ、ヒツジ、ヤギ、フェレット等の家畜又は愛玩動物、そしてヒト、カニクイザル、アカゲザル、コモンマーモセット、ニホンザル、ゴリラ、チンパンジー等の霊長類が挙げられる。特に好ましくはヒトである。 The cell-derived species in the present specification may be a multicellular organism. It is preferably an animal, more preferably a mammal. For example, rodents such as mice, rats, hamsters, guinea pigs, gerbils, livestock or pets such as dogs, cats, rabbits, cows, horses, pigs, sheep, goats, ferrets, and humans, crab monkeys, red-tailed monkeys, common marmosets. , Japanese monkeys, gorillas, chimpanzees and other primates. Especially preferably human.
　本明細書における細胞は、自己、同種異系又は異種の細胞であってよく、好ましくは、同種異系の細胞である。本明細書における細胞は、培養された細胞又は継代された細胞であってよい。 The cells herein may be autologous, allogeneic or heterologous cells, preferably allogeneic cells. The cells herein may be cultured cells or passaged cells.
（３）細胞塊
　本明細書において「細胞塊」とは、細胞凝集によって形成される塊状の凝集体である。細胞凝集塊又はスフェロイドとも呼ばれる。「細胞凝集」とは、複数の細胞が三次元的に集合して、集塊を形成することをいう。異なる細胞による凝集と同一細胞による凝集があるが、本明細書ではいずれであってもよい。同一細胞による凝集は、1つの細胞の増殖によって集塊が形成される場合を含む。細胞凝集の機構としては、限定はしないが、膜タンパク質や細胞膜、細胞外基質を介した細胞間の接着、細胞表面のカドヘリンを介した細胞間の接着等が挙げられる。 (3) Cell mass In the present specification, the “cell mass” is a mass-like aggregate formed by cell aggregation. Also called cell agglutinin or spheroid. "Cell aggregation" means that a plurality of cells are three-dimensionally aggregated to form an agglutination. There are agglutination by different cells and agglutination by the same cell, whichever is used herein. Aggregation by the same cell includes the case where agglomerates are formed by the proliferation of one cell. The mechanism of cell aggregation includes, but is not limited to, adhesion between cells via membrane proteins, cell membranes, extracellular matrix, and adhesion between cells via cadherin on the cell surface.
　細胞塊の形状は、特に限定はしない。細胞凝集によって形成される細胞塊は、通常、略球状を呈することから、略球状が好ましい。 The shape of the cell mass is not particularly limited. Since the cell mass formed by cell aggregation usually has a substantially spherical shape, a substantially spherical shape is preferable.
　細胞塊の大きさは、平均直径が５０μｍ以上、１００μｍ以上、１５０μｍ以上、２００μｍ以上、２２０μｍ以上、２５０μｍ以上、３００μｍ以上、３５０μｍ以上、又は４００μｍ以上であり、７５０μｍ以下、７００μｍ以下、６５０μｍ以下、６００μｍ以下、５５０μｍ以下、５００μｍ以下、又は４５０μｍ以下である。ここで、本明細書において「直径」とは、略球形の細胞塊の直径（μｍ）を意味し、本明細書において「平均直径」とは、複数の細胞塊の直径の相加平均（μｍ）を意味する。前記細胞塊の平均直径の測定は、例えば顕微鏡及びイメージングソフトウェア（例えば、ＯＬＹＭＰＵＳ社製ｃｅｌｌＳｅｎｓｅ）を用いて細胞塊を撮影した像から算出することができる。具体的には、細胞塊の撮影像の任意の３方向から直径を測定し、それらの平均値を算出することにより、細胞塊の平均直径を求めることができる。細胞塊の平均直径が前記範囲内にあるときに、鋳型の内部空間内に細胞塊を配置した際に生じ得る細胞塊同士の接触部の間隙を低減、均一化することが可能となり、その結果、培養工程における細胞培養物の亀裂の発生を防止することができる。そのため、得られる三次元細胞構造体の細胞密度及び形状の均一性を向上させることができる。なお、細胞塊の平均直径が５０μｍ未満であると、得られる三次元細胞構造体の形状が不均一となるため、好ましくない。また、細胞塊の平均直径が７５０μｍより大きいと、鋳型の内部空間内の細胞塊同士の接触部の間隙が大きくなるため、培養工程において細胞塊同士が不均一に融合し、亀裂と粗い表面とを有する細胞培養物が形成されるため、好ましくない。 The size of the cell mass is 50 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, 220 μm or more, 250 μm or more, 300 μm or more, 350 μm or more, or 400 μm or more, and 750 μm or less, 700 μm or less, 650 μm or less, 600 μm. Hereinafter, it is 550 μm or less, 500 μm or less, or 450 μm or less. Here, the “diameter” in the present specification means the diameter (μm) of a substantially spherical cell mass, and the “average diameter” in the present specification is an arithmetic mean (μm) of the diameters of a plurality of cell clusters. ) Means. The measurement of the average diameter of the cell mass can be calculated from an image obtained by photographing the cell mass using, for example, a microscope and imaging software (for example, cellSense manufactured by OLYMPUS). Specifically, the average diameter of the cell mass can be obtained by measuring the diameter from any three directions of the photographed image of the cell mass and calculating the average value thereof. When the average diameter of the cell mass is within the above range, it is possible to reduce and homogenize the gap between the contact portions between the cell masses that may occur when the cell mass is placed in the inner space of the template, and as a result. , It is possible to prevent the occurrence of cracks in the cell culture in the culture step. Therefore, the cell density and shape uniformity of the obtained three-dimensional cell structure can be improved. If the average diameter of the cell mass is less than 50 μm, the shape of the obtained three-dimensional cell structure becomes non-uniform, which is not preferable. Further, when the average diameter of the cell mass is larger than 750 μm, the gap between the contact portions between the cell masses in the inner space of the template becomes large, so that the cell masses are unevenly fused in the culture step, resulting in cracks and a rough surface. It is not preferable because a cell culture having the above is formed.
（４）細胞集団
　本明細書において「細胞集団」とは、少なくとも前記細胞塊を包含する複数の細胞からなる集団をいう。細胞集団は、細胞塊のみで構成されていてもよいし、単一細胞又は数個の細胞を含んでいてもよい。細胞集団を構成する各細胞塊、及び／又は各細胞は、細胞集団中において、細胞接着せずに、培養液等の液体中で互いに分離して存在することができる。この場合の細胞集団を含む液体を本明細書ではしばしば「細胞懸濁液」と表記する。 (4) Cell population As used herein, the term "cell population" refers to a population consisting of a plurality of cells including at least the cell mass. The cell population may consist solely of cell clusters or may include a single cell or several cells. Each cell mass and / or each cell constituting the cell population can exist separately from each other in a liquid such as a culture solution without cell adhesion in the cell population. The liquid containing the cell population in this case is often referred to herein as a "cell suspension."
　細胞集団中に含まれる全細胞及び細胞塊において、前記大きさの範囲内にある細胞塊の比率は、５０％以上、５１％以上、５２％以上、５３％以上、５４％以上、５５％以上、６０％以上、６５％以上、７０％以上、７５％以上、８０％以上、８５％以上、又は９０％以上であることが好ましい。細胞集団が細胞塊を５０％以上含むと、細胞集団の同質性が高まる結果、鋳型の内部空間内の細胞塊の配置がより均一になり、かつ培養工程において細胞塊同士がより均一に融合するため、目視可能な亀裂がなく、かつ形状の均一性が一層向上した三次元細胞構造体を取得することができる。 Among all cells and cell clusters contained in the cell population, the ratio of cell clusters within the above size range is 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more. , 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more. When the cell population contains 50% or more of the cell mass, the homogeneity of the cell population is enhanced, and as a result, the arrangement of the cell mass in the inner space of the template becomes more uniform, and the cell mass fuses more uniformly in the culture step. Therefore, it is possible to obtain a three-dimensional cell structure having no visible cracks and further improved shape uniformity.
　本発明の細胞集団は、２個以上の任意の数の細胞塊を含むことができ、例えば、１．０×１０１個、１．０×１０２個、２．０×１０２個、５．０×１０２個、１．０×１０３個、２．０×１０３個、５．０×１０３個、１．０×１０４個、２．０×１０４個、５．０×１０４個、１．０×１０５個、１．０×１０６個、１．０×１０７個、１．０×１０８個、１．０×１０９個、１．０×１０１０個以上又は以下の細胞塊を含むことができるが、これらに限定されない。 The cell population of the present invention can contain any number of cell clusters of 2 or more, eg, 1.0 × 10 1 , 1.0 × 10 2 , 2.0 × 10 2 , 5, .0 × 10 2 pcs, 1.0 × 10 3 pcs, 2.0 × 10 3 pcs, 5.0 × 10 3 pcs, 1.0 × 10 4 pcs, 2.0 × 10 4 pcs, 5.0 × 10 4 pieces, 1.0 × 10 5 pieces, 1.0 × 10 6 pieces, 1.0 × 10 7 pieces, 1.0 × 10 8 pieces, 1.0 × 10 9 pieces, 1.0 × 10 It can include, but is not limited to, 10 or more or less cell clusters.
　細胞集団に含まれる複数の細胞塊、又は複数の細胞は、互いに同一種由来又は異なる種由来のいずれであってもよい。 The plurality of cell clusters or the plurality of cells contained in the cell population may be derived from the same species or different species from each other.
　本明細書で使用する細胞懸濁液は、細胞以外に任意の成分を含んでいてもよく、例えば、塩類、糖類、タンパク質、アミノ酸、培地成分、緩衝剤等を挙げることができるが、これらに限定されない。また、細胞懸濁液のｐＨは、中性付近のｐＨ、例えば、ｐＨ５．５以上、ｐＨ６．０以上、ｐＨ６．５以上又はｐＨ７．０以上とすることができ、またｐＨ１０．５以下、ｐＨ９．５以下、ｐＨ８．５以下又はｐＨ８．０以下とすることができるが、これらに限定されない。 The cell suspension used in the present specification may contain any component other than cells, and examples thereof include salts, sugars, proteins, amino acids, medium components, buffers, and the like. Not limited. The pH of the cell suspension can be near neutral, for example, pH 5.5 or higher, pH 6.0 or higher, pH 6.5 or higher or pH 7.0 or higher, and pH 10.5 or lower, pH 9 or higher. It can be 5.5 or less, pH 8.5 or less, or pH 8.0 or less, but is not limited thereto.
（５）培地
　本明細書において「培地」とは、細胞を培養するために調製された液状、半固形状又は固形状の物質をいう。原則として、細胞の増殖及び／又は維持に不可欠の成分を必要最小限以上包含する。本明細書で使用する培地は、特に断りがない限り、動物由来細胞の培養に使用する動物細胞用液体培地が該当する。また、培地は、限定はしないが、ＢＭＥ培地、ＢＧＪｂ培地、ＣＭＲＬ１０６６培地、Ｇｌａｓｇｏｗ ＭＥＭ培地、Ｉｍｐｒｏｖｅｄ ＭＥＭ Ｚｉｎｃ Ｏｐｔｉｏｎ培地、ＩＭＤＭ培地（Ｉｓｃｏｖｅ’Ｓ Ｍｏｄｉｆｉｅｄ Ｄｕｌｂｅｃｃｏ’Ｓ Ｍｅｄｉｕｍ）、Ｍｅｄｉｕｍ １９９培地、Ｅａｇｌｅ ＭＥＭ培地、αＭＥＭ培地、ＤＭＥＭ培地（Ｄｕｌｂｅｃｃｏ’Ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’Ｓ Ｍｅｄｉｕｍ）、ハムＦ１０培地、ハムＦ１２培地、ＲＰＭＩ １６４０培地、Ｆｉｓｃｈｅｒ’Ｓ培地、及びこれらの混合培地（例えば、ＤＭＥＭ／Ｆ１２培地（Ｄｕｌｂｅｃｃｏ’Ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’Ｓ Ｍｅｄｉｕｍ／Ｎｕｔｒｉｅｎｔ Ｍｉｘｔｕｒe F－１２ Ｈａｍ））等を使用することができる。ＤＭＥＭ／Ｆ１２培地としては特に、ＤＭＥＭ培地とハムＦ１２培地の重量比を好ましくは６０／４０以上４０／６０以下の範囲、例えば５８／４２、５５／４５、５２／４８、５０／５０、４８／５２、４５／５５、又は４２／５８等で混合した培地を用いる。その他、ヒトｉＰＳ細胞やヒトＥＳ細胞の培養に使用されている培地も好適に使用することもできる。 (5) Medium As used herein, the term "medium" refers to a liquid, semi-solid or solid substance prepared for culturing cells. As a general rule, it contains more than the minimum necessary components essential for cell proliferation and / or maintenance. Unless otherwise specified, the medium used in the present specification corresponds to a liquid medium for animal cells used for culturing animal-derived cells. In addition, the medium is not limited, but is BME medium, BGJb medium, CMRL1066 medium, Glasgo MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscover's Modified Dulvecco'S Medium), Medium Medium 199 medium. αMEM medium, DMEM medium (Dulvecco'S Modified Eagle'S Medium), ham F10 medium, ham F12 medium, RPMI 1640 medium, Fisher'S medium, and a mixed medium thereof (for example, DMEM / F12 medium (Dulvecco'S Medium)). Eagle'S Medium / Nutrition Mixture F-12 Ham)) and the like can be used. As the DMEM / F12 medium, the weight ratio of the DMEM medium and the ham F12 medium is preferably in the range of 60/40 or more and 40/60 or less, for example, 58/42, 55/45, 52/48, 50/50, 48 /. A medium mixed with 52, 45/55, 42/58, etc. is used. In addition, the medium used for culturing human iPS cells and human ES cells can also be preferably used.
　本発明で使用する培地は、血清を含有した培地であってもよく、無血清培地であってもよい。血清は、限定はしないが、例えばウシ胎児血清（ＦＢＳ）、ウマ血清等である。血清を含有した培地において、培地中の血清の終濃度は、１％以上、２％以上、３％以上、４％以上又は５％以上であり、また２０％以下、１８％以下、１６％以下、１４％以下、１２％以下又は１０％以下であり、さらに好ましくは９％以下であり、さらに好ましくは８％以下であればよい。また、無血清培地は、限定はしないが、例えば、ＳＴＫ１やＳＴＫ２（ＤＳファーマバイオメディカル社製）、ＥＸＰＲＥＰ　ＭＳＣ　Ｍｅｄｉｕｍ（バイオミメティクスシンパシーズ社製）、Ｃｏｒｎｉｎｇ　ｓｔｅｍｇｒo　ヒト接着性幹細胞培地（コーニング社製）等の市販の無血清培地であってよい。 The medium used in the present invention may be a medium containing serum or a serum-free medium. Serum is, but is not limited to, fetal bovine serum (FBS), horse serum and the like. In the medium containing serum, the final concentration of serum in the medium is 1% or more, 2% or more, 3% or more, 4% or more or 5% or more, and 20% or less, 18% or less, 16% or less. , 14% or less, 12% or less or 10% or less, more preferably 9% or less, still more preferably 8% or less. The serum-free medium is not limited, but is, for example, STK1 or STK2 (manufactured by DS Pharma Biomedical), EXPREP MSC Medium (manufactured by Biomimetic Sympathies), Corning stemgro human adhesive stem cell medium (manufactured by Corning), etc. It may be a commercially available serum-free medium.
　本明細書で使用する培地は様々な培養添加物を加えて、各種細胞に特異的な培地に調製することもできる。「培養添加物」とは、培養目的で培地に添加される物質である。培養添加物の具体例として、限定はしないが、血清、血清代替試薬、インスリン、トランスフェリン、セレン、サイトカイン、増殖因子、アルブミン、炭酸水素ナトリウム、脂肪酸、アミノ酸（例えば、非必須アミノ酸）、Ｌ－アスコルビン酸、ビタミン、抗酸化剤、２－メルカプトエタノール、ピルビン酸、緩衝剤、無機塩類、多糖類、抗生剤等が挙げられる。インスリン、トランスフェリン、及びサイトカインは、動物（好ましくは、ヒト、マウス、ラット、ウシ、ウマ、ヤギ等）の組織又は血清等から分離した天然由来のものであってもよいし、遺伝子工学的に作製した組換えタンパク質であってもよい。また、増殖因子は、限定はしないが、例えば、ＦＧＦ２（Ｂａｓｉｃ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ－２）、ＥＧＦ（Ｅｐｉｄｅｒｍａｌ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ）、ＴＧＦ－β１（Ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ－β１）、ＶＥＧＦ（Ｖａｓｃｕｌａｒ　ｅｎｄｏｔｈｅｌｉａｌ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ）、ＰＤＧＦ（Ｐｌａｔｅｌｅｔ－ｄｅｒｉｖｅｄ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ）、Ａｃｔｉｖｉｎ Ａ、ＩＧＦ－１、ＭＣＰ－１、ＩＬ－６、ＰＡＩ、ＰＥＤＦ、ＩＧＦＢＰ－２、ＬＩＦ及びＩＧＦＢＰ－７を使用することができる。抗生剤は、限定するものではないが、例えば、ペニシリン、ストレプトマイシン、アンホテリシンＢ等を使用することができる。本発明で用いる培地の培養添加物として、特に好ましい増殖因子は、ＦＧＦ２である。培養添加物は、培地に１種以上含むことができる。 The medium used in the present specification can be prepared as a medium specific to various cells by adding various culture additives. A "culture additive" is a substance added to a medium for the purpose of culturing. Specific examples of culture additives include, but are not limited to, serum, serum substitute reagents, insulin, transferrin, selenium, cytokines, growth factors, albumin, sodium hydrogen carbonate, fatty acids, amino acids (eg, non-essential amino acids), L-ascorbin. Examples include acids, vitamins, antioxidants, 2-mercaptoethanol, pyruvate, buffers, inorganic salts, polysaccharides, antibiotics and the like. Insulin, transferrin, and cytokines may be of natural origin isolated from animal (preferably human, mouse, rat, bovine, horse, goat, etc.) tissues or sera, or genetically engineered. It may be a recombinant protein. The growth factors are not limited, but are, for example, FGF2 (Basic fiberblast growth factor-2), EGF (Epidermal growth factor), TGF-β1 (Transforming growth factor-β1), VEGF (VEGF). (Platelet-developed growth factor), Activin A, IGF-1, MCP-1, IL-6, PAI, PEDF, IGFBP-2, LIF and IGFBP-7 can be used. Antibiotics include, but are not limited to, penicillin, streptomycin, amphotericin B and the like. A particularly preferred growth factor as a culture additive for the medium used in the present invention is FGF2. One or more culture additives can be contained in the medium.
　培養添加物は、溶液、誘導体、塩又は混合試薬等の形態で培地に添加することができる。例えば、Ｌ－アスコルビン酸は、２－リン酸アスコルビン酸マグネシウム等の誘導体の形態で培地に添加してもよく、セレンは亜セレン酸塩（亜セレン酸ナトリウム等）の形態で培地に添加してもよい。また、インスリン、トランスフェリン、及びセレンに関しては、ＩＴＳ試薬（インスリン-トランスフェリン-セレン）の形態で培地に添加することもできる。また、Ｌ－アスコルビン酸、インスリン、トランスフェリン、セレン及び炭酸水素ナトリウムから選択される少なくとも１つが既に添加された市販の培地を使用することもできる。インスリン及びトランスフェリンを添加した市販の培地としては、ＣＨＯ－Ｓ－ＳＦＭ II（ライフテクノロジーズジャパン株式会社）、Ｈｙｂｒｉｄｏｍａ－ＳＦＭ（ライフテクノロジーズジャパン株式会社）、ｅＲＤＦ Ｄｒｙ Ｐｏｗｄｅｒｅｄ Ｍｅｄｉａ（ライフテクノロジーズジャパン株式会社）、ＵｌｔｒａＣＵＬＴＵＲＥＴＭ（ＢｉｏＷｈｉｔｔａｋｅｒ社）、ＵｌｔｒａＤＯＭＡＴＭ（ＢｉｏＷｈｉｔｔａｋｅｒ社）、ＵｌｔｒａＣＨＯＴＭ（ＢｉｏＷｈｉｔｔａｋｅｒ社）、ＵｌｔｒａＭＤＣＫＴＭ（ＢｉｏＷｈｉｔｔａｋｅｒ社）、ＳＴＥＭＰＲＯ（登録商標） ｈＥＳＣ ＳＦＭ（ライフテクノロジーズジャパン株式会社）、ｍＴｅＳＲ１（Ｖｅｒｉｔａｓ社）、及びＴｅＳＲ２（Ｖｅｒｉｔａｓ社）等が挙げられる。 Culture additives can be added to the medium in the form of solutions, derivatives, salts, mixed reagents and the like. For example, L-ascorbic acid may be added to the medium in the form of a derivative such as magnesium 2-ascorbic acid, and selenium may be added to the medium in the form of a selenium salt (sodium selenite, etc.). May be good. Insulin, transferrin, and selenium can also be added to the medium in the form of an ITS reagent (insulin-transferrin-selenium). It is also possible to use a commercially available medium to which at least one selected from L-ascorbic acid, insulin, transferrin, selenium and sodium hydrogen carbonate has already been added. Commercially available media containing insulin and transferase include CHO-S-SFM II (Life Technologies Japan Co., Ltd.), Hybridoma-SFM (Life Technologies Japan Co., Ltd.), eRDF Dry Powered Media (Life Technologies Japan Co., Ltd.), and UltraCULTURE. TM (BioWittaker), UltraDOMA TM (BioWhittaker), UltraCHO TM (BioWhittaker), UltraMDCK TM (BioWhiteker), STEMPRO (Registered Trademarks) (Veritas) and the like.
（６）鋳型
　本明細書において「鋳型」とは、三次元細胞構造体を製造するための成形用の型をいう。鋳型は、その性質上、内部に空間（キャビティ：本明細書では、「内部空間」と換言し、表記する場合がある）を有し、その内部空間は、通常、三次元細胞構造体の外観を呈する形状を有する。 (6) Mold As used herein, the term "template" refers to a molding mold for producing a three-dimensional cell structure. Due to its nature, the template has an internal space (cavity: sometimes referred to as "internal space" in the present specification), and the internal space is usually the appearance of a three-dimensional cell structure. It has a shape that exhibits.
　鋳型の外部形状は、特に制限はされない。製造する三次元細胞構造体に相似な形状、又は非相似な形状のいずれであってもよい。一方、内部空間の形状は、製造する三次元細胞構造体の外部形状と全部又は一部が相似な形状を有する。それ故に、内部空間の形状は、三次元細胞構造体の外部形状に合わせて、所望の形状とすることができる。例えば、円筒形の三次元細胞構造体の製造を所望する場合、鋳型の内部空間を円筒状にすればよい。一般に、鋳型の壁部が薄い場合には、外部形状と内部空間の形状が相似する。そのため、外部形状も製造する三次元細胞構造体の相似形状に近づく。 The outer shape of the mold is not particularly limited. It may have a shape similar to or dissimilar to the three-dimensional cell structure to be produced. On the other hand, the shape of the internal space has a shape similar to the external shape of the three-dimensional cell structure to be manufactured, in whole or in part. Therefore, the shape of the internal space can be made into a desired shape according to the external shape of the three-dimensional cell structure. For example, if it is desired to produce a cylindrical three-dimensional cell structure, the internal space of the template may be cylindrical. Generally, when the wall portion of the mold is thin, the outer shape and the shape of the inner space are similar. Therefore, it approaches the similar shape of the three-dimensional cell structure that also manufactures the outer shape.
　鋳型は、内部空間内に配置される内壁部（コア）を有することができる。この場合、鋳型において、内部空間が凹部（雌型）、内壁部が凸部（雄型）に相当し、内部空間と内壁部との間に形成される空洞部が、目的とする三次元細胞構造体の形状を呈する。内壁部の形状は、三次元細胞構造体の外部又は内部形状に合わせて、所望の形状とすることができる。例えば、ドーナツ状又は管状の三次元細胞構造体の製造を所望する場合、鋳型の内部空間を円筒状に、また内壁部を内部空間よりも小さい円柱状にすればよい。内部空間と内壁部の間に形成されるドーナツ状又は管状の空洞部内に充填する細胞集団の分量を調整することで、ドーナツ状又は管状の三次元細胞構造体を製造することができる。 The mold can have an inner wall portion (core) arranged in the internal space. In this case, in the template, the internal space corresponds to a concave portion (female type) and the inner wall portion corresponds to a convex portion (male type), and the hollow portion formed between the internal space and the inner wall portion is the target three-dimensional cell. It exhibits the shape of a structure. The shape of the inner wall portion can be a desired shape according to the outer or inner shape of the three-dimensional cell structure. For example, when it is desired to produce a donut-shaped or tubular three-dimensional cell structure, the internal space of the template may be cylindrical and the inner wall may be cylindrical, which is smaller than the internal space. A donut-shaped or tubular three-dimensional cell structure can be produced by adjusting the amount of the cell population to be filled in the donut-shaped or tubular cavity formed between the internal space and the inner wall portion.
　鋳型の素材は、壁部の全部又は一部が通液可能な多孔構造を有し得る材質であればよく、特に限定はしない。後述するように鋳型は培地内に浸漬されるため、非水溶性素材又は非易溶性素材であることが望ましい。「非水溶性素材」とは、水又は水溶液に溶解しない素材をいう。無生物系、生物系、及びそれらの混合物が含まれる。非無生物系の非水溶性素材とは、素材が直接的な生物由来ではない非水溶性素材をいう。例えば、プラスチック（化学繊維を含む）、ガラス、金属、シリコン、合成ゴム、セラミックス等が挙げられる。プラスチックであれば、例えば、ポリエチレン（ＰＥ）、ポリエチレンテレフタレート（ＰＥＴ）、ポリプロピレン（ＰＰ）、ポリスチレン（ＰＳ）、ポリウレタン（ＰＵ）、ポリ塩化ビニル（ＰＶＣ）、ポリ塩化ビニリデン（ＰＶＤＣ）、ポリカーボネート（ＰＣ）、ポリサルフォン（ＰＳＵ）、ポリアリレート（ＰＡＲ）、ポリアミド（ナイロン）、ポリビニルアルコール（ＰＶＡ）等を利用することができる。金属であれば、例えば、金（Ａｕ）、白金（Ｐｔ）、銀（Ａｇ）、銅（Ｃｕ）、ニッケル（Ｎｉ）、チタン（Ｔｉ）、アルミニウム（Ａｌ）、タルタル（Ｔａ）等の純金属の他、ステンレス（ＳＵＳ）、コバルト合金、ニチノール（ＮｉＴｉ）等の合金が挙げられる。生物系の非水溶性素材とは、生物によって直接生産された非水溶性素材をいう。例えば、天然繊維（セルロース、ケラチン、フィブロイン等）、天然樹脂(例えば、天然ゴム又は漆）、骨（サンゴ骨格、海綿骨格等を含む）、歯、角、木質部、及び木炭等が挙げられる。「非易溶性性素材」とは、常温常圧下では水又は水溶液に溶解しない素材をいう。限定はしないが、通常は生物由来である。例えば、多糖類高分子（例えば、寒天、マンナン）、ゲル化タンパク質（例えば、ゼラチン、コラーゲン）、又はそれらの混合物が挙げられる。 The material of the mold may be any material as long as it can have a porous structure through which all or part of the wall can pass, and is not particularly limited. Since the template is immersed in the medium as described later, it is desirable that the template is a water-insoluble material or a non-soluble material. "Water-insoluble material" means a material that is insoluble in water or an aqueous solution. Includes inanimate systems, biological systems, and mixtures thereof. An inanimate water-insoluble material is a water-insoluble material whose material is not directly derived from a living organism. For example, plastic (including chemical fibers), glass, metal, silicon, synthetic rubber, ceramics and the like can be mentioned. For plastics, for example, polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), polyurethane (PU), polyvinylidene chloride (PVC), polyvinylidene chloride (PVDC), polycarbonate (PC). ), Polysulfone (PSU), polyarylate (PAR), polyamide (nylon), polyvinyl alcohol (PVA) and the like can be used. If it is a metal, for example, a pure metal such as gold (Au), platinum (Pt), silver (Ag), copper (Cu), nickel (Ni), titanium (Ti), aluminum (Al), tartar (Ta), etc. Other examples include alloys such as stainless steel (SUS), cobalt alloys, and nitinol (NiTi). Biological water-insoluble materials refer to water-insoluble materials produced directly by living organisms. Examples thereof include natural fibers (cellulose, keratin, fibroin, etc.), natural resins (eg, natural rubber or lacquer), bones (including coral skeleton, spongy skeleton, etc.), teeth, horns, xylem, charcoal, and the like. The "non-soluble material" means a material that does not dissolve in water or an aqueous solution under normal temperature and pressure. Although not limited, it is usually of biological origin. Examples include polysaccharide macromolecules (eg, agar, mannan), gelled proteins (eg, gelatin, collagen), or mixtures thereof.
　鋳型は二以上の異なる素材で構成された複合素材であってもよい。例えば、壁部において、鋳型外部に面した素材と内部空間に面した素材を異なる素材とする場合が挙げられる。このような構造は、壁部を構成する各面が異なる環境に配置される場合等で、その環境に合わせて適切な素材を用いることができるので便利である。例えば、内部空間壁面は、細胞直接に接触する細胞接触面である。この場合、内部空間壁面は細胞非接着素材であることが好ましい。細胞接触面が細胞接着性の高い素材の場合、製造後の三次元細胞構造体と鋳型との分離が困難になるためである。細胞非接着素材としては、例えば、ステンレス、アルミニウム、ポリプロピレン等が挙げられる。また、内部空間壁面として、任意の素材の表面に、細胞接着を阻害する物質をコーティングしたものを採用することもできる。細胞接着を阻害する物質としては、例えば、寒天、ポリ（２－ヒドロキシエチルメタクリレート）、ポリエチレングリコール等が挙げられるが、これらに限定されない。 The mold may be a composite material composed of two or more different materials. For example, in the wall portion, a material facing the outside of the mold and a material facing the internal space may be different materials. Such a structure is convenient because when each surface constituting the wall portion is arranged in a different environment, an appropriate material can be used according to the environment. For example, the wall surface of the interior space is a cell contact surface that comes into direct contact with cells. In this case, the inner space wall surface is preferably made of a non-cell adhesive material. This is because when the cell contact surface is a material having high cell adhesion, it becomes difficult to separate the three-dimensional cell structure and the template after production. Examples of the cell non-adhesive material include stainless steel, aluminum, polypropylene and the like. Further, as the internal space wall surface, a material having a surface coated with a substance that inhibits cell adhesion can be adopted. Examples of the substance that inhibits cell adhesion include, but are not limited to, agar, poly (2-hydroxyethyl methacrylate), polyethylene glycol, and the like.
　本発明で使用する鋳型の特徴として、前述のように壁部の全部又は一部に通液可能な多孔構造を有する。「壁部」とは、鋳型における内部空間の壁面（側面、底面、及び上面を含む）を構成する部分をいう。また「通液可能」とは、液体が壁部を通過可能なことをいう。壁部におけるこの通液性により、液体は、弊部外部と内部空間とを自由に往来できる。ここでいう「液体」は、限定はしないが、主に水又は水溶液である。水溶液は、培地やバッファを包含する。バッファとしては、ダルベッコリン酸バッファ（ＤＰＢＳ）、アール平衡塩溶液（ＥＢＳＳ）、ハンクス平衡塩溶液（ＨＢＳＳ）、リン酸バッファ（ＰＢＳ）等を用いることができるが、これらに限定されない。 As a feature of the mold used in the present invention, it has a porous structure that allows liquid to pass through all or part of the wall portion as described above. "Wall portion" refers to a portion of the mold that constitutes the wall surface (including the side surface, bottom surface, and top surface) of the internal space. Further, "liquid passable" means that the liquid can pass through the wall portion. Due to this liquid permeability in the wall part, the liquid can freely move between the outside and the inside space of the part. The "liquid" referred to here is mainly water or an aqueous solution, although it is not limited. Aqueous solution includes medium and buffer. As the buffer, a darbecolinic acid buffer (DPBS), an Earl balanced salt solution (EBSS), a Hanks balanced salt solution (HBSS), a phosphate buffer (PBS) and the like can be used, but the buffer is not limited thereto.
　本明細書において「多孔構造」とは、複数の孔を有する構造をいう。孔の全部又は一部は多孔構造を有する素材表面に開孔している。多孔構造の例として、メッシュ構造や気泡構造が表面開孔した構造等が挙げられる。多孔構造における孔の形状は、限定はしない。円形、略円形、楕円形、多角形、略多角形、不定形、又はそれらの組み合わせのいずれであってもよい。個々の孔サイズは、上限は前記細胞塊が通過できない大きさで、かつ細胞集団を鋳型内部空間に積層した際のそれらの支持、及び鋳型の自立が可能な大きさが好ましい。また、下限は孔が目詰まりを生じることなく通液性を保持できる大きさが好ましい。例えば、前記細胞塊の平均直径を「ｄ」、孔の長径及び／又は最大対角線を「Ｄ」としたときに、前記細胞塊の平均直径に対する孔の長径及び／又は最大対角線の比率（ｄ／Ｄ）が１．０より大、１．１以上、１．２以上、１．３以上、１．４以上、１．５以上、又は２．０以上であり、また３．０以下、２．９以下、２．８以下、２．９以下、２．６以下、又は２．５以下であればよい。比率（ｄ／Ｄ）が１．０以下であると、鋳型内部空間に配置した細胞塊が孔を通過してしまう虞があるため、好ましくない。また、比率（ｄ／Ｄ）が３．０より大きいと、鋳型内部空間に細胞塊を配置した際に、細胞塊によって孔が目詰まりを生じ、十分な通液性を確保できない虞があるため、好ましくない。具体的な孔サイズの例として、孔の長径及び／又は最大対角線が５０μｍ以上、１００μｍ以上、１５０μｍ以上、２００μｍ以上、２２０μｍ以上、２５０μｍ以上、又は３００μｍ以上、そして７５０μｍ以下、７００μｍ以下、６５０μｍ以下、６００μｍ以下、５５０μｍ以下、５００μｍ以下、又は４５０μｍ以下である。 In the present specification, the "porous structure" means a structure having a plurality of pores. All or part of the holes are open on the surface of the material having a porous structure. Examples of the porous structure include a mesh structure and a structure in which a bubble structure has a perforated surface. The shape of the pores in the porous structure is not limited. It may be circular, substantially circular, elliptical, polygonal, substantially polygonal, amorphous, or a combination thereof. It is preferable that the upper limit of the individual pore size is a size that the cell mass cannot pass through, and that the cell population can be supported when the cell population is laminated in the template internal space and the template can be self-supporting. Further, the lower limit is preferably a size capable of maintaining liquid permeability without causing clogging of the holes. For example, when the average diameter of the cell mass is "d" and the major axis and / or the maximum diagonal of the pore is "D", the ratio of the major axis and / or the maximum diagonal of the pore to the average diameter of the cell mass (d /). D) is greater than 1.0, 1.1 or more, 1.2 or more, 1.3 or more, 1.4 or more, 1.5 or more, or 2.0 or more, and 3.0 or less, 2. It may be 9 or less, 2.8 or less, 2.9 or less, 2.6 or less, or 2.5 or less. If the ratio (d / D) is 1.0 or less, the cell mass arranged in the mold internal space may pass through the pores, which is not preferable. Further, if the ratio (d / D) is larger than 3.0, when the cell mass is arranged in the template internal space, the pores may be clogged by the cell mass, and sufficient liquid permeability may not be ensured. , Not preferable. As an example of a specific hole size, the major axis and / or the maximum diagonal of the hole is 50 μm or more, 100 μm or more, 150 μm or more, 200 μm or more, 220 μm or more, 250 μm or more, or 300 μm or more, and 750 μm or less, 700 μm or less, 650 μm or less, It is 600 μm or less, 550 μm or less, 500 μm or less, or 450 μm or less.
　鋳型壁部の多孔構造領域における開孔率とは、孔部分の全体面積に占める割合を意味する。例えばメッシュ構造等の場合、孔部分の平均直径を「Ａ」、線径を「ａ」としたときに、（Ａ／Ａ＋ａ）２×１００（％）となる。本明細書における前記開孔率は、４０％以上、４５％以上、５０％以上、５５％以上、６０％以上、６５％以上、又は７０％以上であり、また９０％以下、８５％以下、８０％以下、又は７５％以下であることが好ましい。開孔率を４０％以上、９０％以下とすることにより、内部空間内の細胞集団に対する栄養供給と支持を確保でき、生存状態を維持したまま三次元細胞構造体を形成することができる。開孔率が４０％未満であると、静置培養時において内部空間への十分な通液性を保持できず、内部空間内の細胞集団に対して栄養供給を確保できなくなるため、好ましくない。また、開孔率が９０％より大きいと、内部空間内の細胞集団を支持するための鋳型強度が不足する可能性があるため、好ましくない。 The pore opening ratio in the porous structure region of the mold wall portion means the ratio of the pore portion to the total area. For example, in the case of a mesh structure or the like, when the average diameter of the hole portion is “A” and the wire diameter is “a”, it becomes (A / A + a) 2 × 100 (%). The aperture ratio in the present specification is 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more, and 90% or less, 85% or less, It is preferably 80% or less, or 75% or less. By setting the aperture ratio to 40% or more and 90% or less, it is possible to secure the nutrient supply and support for the cell population in the internal space, and to form a three-dimensional cell structure while maintaining the viable state. If the aperture ratio is less than 40%, sufficient fluid permeability to the internal space cannot be maintained during static culture, and the nutrient supply to the cell population in the internal space cannot be secured, which is not preferable. Further, if the aperture ratio is larger than 90%, the template strength for supporting the cell population in the internal space may be insufficient, which is not preferable.
　前述の内壁部の素材や特徴は、鋳型のそれらと同一であってもよいし、全部又は一部が異なっていてもよい。例えば、内壁部の細胞接着面は、鋳型と同様に細胞非接着素材であることが好ましい。一方、内壁部は、通液性がなくてもよいし、あるいは鋳型と同様に通液性を有する多孔構造であってもよい。通液性を有する場合、具体的な多孔構造は鋳型と同じであってもよいし、異なっていてもよい。例えば、鋳型壁部側面がメッシュ構造で、内壁部が気泡構造を有する多孔構造の場合が挙げられる。 The material and characteristics of the inner wall portion described above may be the same as those of the mold, or may be completely or partially different. For example, the cell adhesion surface of the inner wall portion is preferably a cell non-adhesion material as in the template. On the other hand, the inner wall portion may not have liquid permeability, or may have a porous structure having liquid permeability similar to the mold. When it has liquid permeability, the specific porous structure may be the same as or different from that of the mold. For example, there is a case where the side surface of the mold wall portion has a mesh structure and the inner wall portion has a porous structure having a bubble structure.
　上記構成を有する鋳型（内壁部を含む）は、本発明の三次元細胞構造体の製造方法を具現化可能な鋳型、すなわち三次元細胞構造体製造用鋳型として利用することもできる。 The template having the above structure (including the inner wall portion) can also be used as a template capable of embodying the method for producing a three-dimensional cell structure of the present invention, that is, a template for producing a three-dimensional cell structure.
１－３．方法
　本発明の三次元細胞構造体の製造方法のフローを図１に示す。本発明の製造方法は、細胞集団配置工程（Ｓ０１０２)、及び培養工程（Ｓ０１０３)を必須の工程として、また、細胞集団調製工程（Ｓ０１０１)、及び分離工程（Ｓ０１０４）を選択工程として含む。以下、各工程について、具体的に説明をする。 1-3. Method The flow of the method for producing a three-dimensional cell structure of the present invention is shown in FIG. The production method of the present invention includes a cell population arrangement step (S0102) and a culture step (S0103) as essential steps, and a cell population preparation step (S0101) and a separation step (S0104) as selection steps. Hereinafter, each step will be specifically described.
（１）細胞集団調製工程
　「細胞集団調製工程」（Ｓ０１０１)は、平均直径が５０μｍ以上、７５０μｍ以下の細胞塊を含む細胞集団を調製する工程である。本工程は、細胞集団配置工程に先立ち実施される選択的工程である。 (1) Cell Population Preparation Step The “cell population preparation step” (S0101) is a step of preparing a cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less. This step is a selective step carried out prior to the cell population placement step.
　細胞集団の調製方法は特に限定はしない。基本的には当該分野で公知の細胞懸濁液を調製する方法に基づいて、所定の大きさの細胞塊が細胞懸濁液中に含まれるように調製すればよい。細胞塊を含む細胞懸濁液の調製方法には、例えば、接着培養法で得られた細胞、又は生体等より採取した組織片を、例えばＰｒｉｍｅＳｕｒｆａｃｅ（登録商標）プレート（住友ベークライト社製）等の細胞非接触基材上で培養する方法がある。その際、前記基材の表面に細胞同士が接触し続けるような条件で播種することで自発的に細胞が凝集し、細胞塊を形成することができる。細胞塊を含む細胞懸濁液のその他の調製方法としては、細胞懸濁液の液滴を吊り下げる形で維持し、液滴の中で細胞が凝集するハンギングドロップ法、化学的処理及び／又は物理的処理によって調製する方法、及び浮遊培養法によって調製する方法等がある。 The method for preparing the cell population is not particularly limited. Basically, it may be prepared so that a cell mass having a predetermined size is contained in the cell suspension based on a method for preparing a cell suspension known in the art. As a method for preparing a cell suspension containing a cell mass, for example, a cell obtained by an adhesive culture method or a tissue piece collected from a living body or the like is used, for example, a Prime Surface (registered trademark) plate (manufactured by Sumitomo Bakelite Co., Ltd.) or the like. There is a method of culturing on a cell non-contact substrate. At that time, by seeding under the condition that the cells keep in contact with each other on the surface of the base material, the cells can spontaneously aggregate and form a cell mass. Other methods of preparing cell suspensions containing cell clusters include hanging drop methods, chemical treatments and / or methods in which the droplets of the cell suspension are maintained in a suspended form and the cells aggregate in the droplets. There are a method of preparing by physical treatment, a method of preparing by suspension culture method, and the like.
　本明細書において「接着培養法」とは、細胞を培養容器等に接着させて、原則単層で増殖させる培養方法をいう。前記「化学的処理」とは、接着培養法により得られた単層細胞や組織片を、接着細胞の剥離や組織の細胞分散に使用される細胞剥離剤で化学的に処理することをいう。細胞剥離剤としては、トリプシン、コラゲナーゼ、ディスパーゼ、エチレンジアミン四酢酸（ＥＤＴＡ）等を使用することができるが、特に限定されない。細胞剥離剤として、市販の細胞剥離剤を用いてもよい。例えば、トリプシン－ＥＤＴＡ溶液（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）、ＴｒｙｐＬＥ　Ｓｅｌｅｃｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製）、Ａｃｃｕｔａｓｅ（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）、Ａｃｃｕｍａｘ（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社製）等が挙げられるが、これらに限定されない。 In the present specification, the "adhesive culture method" refers to a culture method in which cells are adhered to a culture vessel or the like and, in principle, are grown in a single layer. The "chemical treatment" refers to chemically treating monolayer cells and tissue pieces obtained by the adhesion culture method with a cell release agent used for detachment of adherent cells and cell dispersion of tissues. As the cell exfoliating agent, trypsin, collagenase, dispase, ethylenediaminetetraacetic acid (EDTA) and the like can be used, but the cell exfoliating agent is not particularly limited. As the cell exfoliating agent, a commercially available cell exfoliating agent may be used. For example, trypsin-EDTA solution (manufactured by Thermo Fisher Scientific), TrypLE Select (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Thermo Fisher Scientific), Accutase (manufactured by Stemcell Technology), etc.
　また「物理的処理」とは、化学処理等により細胞接着が部分的に分離された単相細胞や組織片に剥離処理や懸濁処理等の機械的外力を加えることで細胞接着の分離を促進させる処理をいう。化学的処理及び／又は物理的処理による細胞集団の調製方法では、細胞の一部が前記平均直径の細胞塊として細胞懸濁液中に残存するように、細胞を分散する際の酵素量、反応温度、及び反応時間等や、外力の付加強度や付加回数等を適宜勘案し、調製しなければならない。 In addition, "physical treatment" promotes the separation of cell adhesion by applying a mechanical external force such as peeling treatment or suspension treatment to monophasic cells or tissue pieces whose cell adhesion is partially separated by chemical treatment or the like. The process of causing. In the method for preparing a cell population by chemical treatment and / or physical treatment, the amount of enzyme and reaction when dispersing cells so that a part of the cells remains in the cell suspension as a cell mass having the average diameter. It must be prepared in consideration of temperature, reaction time, etc., additional strength of external force, number of additions, etc. as appropriate.
　また本明細書において「浮遊培養法」とは、細胞培養方法の一つで、培地中で細胞を浮遊状態で増殖させる方法をいう。この方法では、培養細胞は培養液中で凝集した細胞塊として存在する。したがって、所定の大きさの細胞塊を調製する場合、浮遊培養法は特に好ましい。浮遊培養法では、細胞内のミオシンシグナル伝達経路に関与するシグナル伝達因子の活性を阻害又は抑制する薬剤を培地中に投与することで細胞の凝集が促進され、逆に同因子の活性を促進又は増幅する薬剤を培地中に投与することで細胞凝集が抑制され得る。これらの作用を利用することで浮遊培養細胞の凝集の促進及び抑制を制御することができ、また、それにより平均直径が５０μｍ以上、７５０μｍ以下の細胞塊を製造することも可能となる。浮遊培養法により所望の大きさの細胞塊を調製する具体的な方法の例として、限定はしないが、例えば、国際公開番号ＷＯ２０１６／１２１７３７、特開２０１９-１１８２７９、国際公開番号ＷＯ２０１９／１３１９４１、及び国際公開番号ＷＯ２０１９／１３１９４２、に開示の方法を参考にすることができる。調製後、細胞集団をバッファや培地で洗浄後、それらに懸濁して細胞懸濁液を得てもよいし、調製した培養液をそのまま細胞懸濁として使用してもよい。 Further, in the present specification, the "suspension culture method" is one of the cell culture methods, and refers to a method in which cells are grown in a floating state in a medium. In this method, the cultured cells exist as a cluster of cells aggregated in the culture medium. Therefore, when preparing a cell mass of a predetermined size, the suspension culture method is particularly preferable. In the suspension culture method, cell aggregation is promoted by administering a drug that inhibits or suppresses the activity of a signal transduction factor involved in the intracellular myosin signal transduction pathway into the medium, and conversely, the activity of the same factor is promoted or Cell aggregation can be suppressed by administering the amplifying agent in the medium. By utilizing these actions, it is possible to control the promotion and suppression of aggregation of suspended cultured cells, and it is also possible to produce cell clusters having an average diameter of 50 μm or more and 750 μm or less. Examples of specific methods for preparing a cell mass of a desired size by the suspension culture method include, but are not limited to, for example, International Publication No. WO2016 / 121737, Japanese Patent Application Laid-Open No. 2019-118279, International Publication No. WO2019 / 131941 and. The method of disclosure can be referred to in International Publication No. WO 2019/131942. After the preparation, the cell population may be washed with a buffer or a medium and then suspended in them to obtain a cell suspension, or the prepared culture solution may be used as it is as a cell suspension.
（２）細胞集団配置工程
　「細胞集団配置工程」（Ｓ０１０２）は、細胞塊を含む細胞集団を、鋳型の内部空間内に配置する工程である。 (2) Cell Population Arrangement Step The “cell population arrangement step” (S0102) is a step of arranging a cell population including a cell mass in the internal space of the template.
　本工程において「配置」とは、細胞塊を含む細胞集団を鋳型の内部空間内、又は内部空間に内壁部を配置した際に形成される空洞部内に充填することをいう。鋳型に配置される前の細胞集団は、細胞塊や細胞が溶液中で分散した細胞懸濁液として存在している。しかし、本工程で鋳型内部に配置されることによって細胞集団に含まれる細胞塊や細胞が集合し、互いに接触することで所望する三次元細胞構造体に成形される。 In this step, "arrangement" means filling the cell population including the cell mass into the inner space of the template or the cavity formed when the inner wall portion is arranged in the inner space. The cell population before being placed in the template exists as a cell mass or a cell suspension in which cells are dispersed in a solution. However, by arranging the cells inside the template in this step, the cell clusters and cells contained in the cell population gather, and when they come into contact with each other, they are formed into a desired three-dimensional cell structure.
　鋳型内部空間内への配置方法は限定しない。例えば、細胞懸濁液を鋳型内部空間内に緩やかに流入すればよい。細胞懸濁液の流入は、鋳型を培地に浸漬した状態で行うこともできる。細胞懸濁液は、前記細胞集団調製工程で調製された細胞懸濁液の他、所定の大きさの細胞塊を含む市販の細胞懸濁液を利用することもできる。 The placement method in the mold internal space is not limited. For example, the cell suspension may slowly flow into the template internal space. The inflow of the cell suspension can also be carried out with the template immersed in the medium. As the cell suspension, in addition to the cell suspension prepared in the cell population preparation step, a commercially available cell suspension containing a cell mass having a predetermined size can also be used.
　鋳型内部空間内への細胞集団の充填量は特に限定はしない。鋳型内部空間と目的の三次元細胞構造体の形状、及び大きさ等を勘案して適宜定めればよい。本明細書において「充填」とは、鋳型の内部に細胞集団を投入した後、静置し、自然沈降により最密になった状態を意味する。本工程の場合、例えば、鋳型内部空間の体積の６０％以上、６５％以上、７０％以上、７５％以上、８０％以上、又は８５％以上、９０％以下、９１％以下、９２％以下、９３％以下、９４％以下、又は９５％以下まで充填すればよい。鋳型内部空間の体積の６０％以上、９５％以下となるよう充填することにより、三次元細胞構造体を製造する際の歩留りを一層向上することができる。一方、充填率が鋳型内部空間の体積の６０％未満の場合には、細胞塊が低密度に存在することにより細胞塊同士の接触部の間隙が大きくなり、培養工程において亀裂と粗い表面とを有する細胞培養物が形成される虞があるため、好ましくない。また、充填率が鋳型内部空間の体積の９５％より大きい場合には、細胞塊が超高密度に存在することにより細胞塊同士の接触による物理的負荷が大きくなるため、好ましくない。 The amount of cell population filled in the mold internal space is not particularly limited. It may be appropriately determined in consideration of the mold internal space, the shape and size of the target three-dimensional cell structure, and the like. As used herein, the term "filling" means a state in which a cell population is placed inside a template, then allowed to stand, and the cells become close-packed by natural sedimentation. In the case of this step, for example, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or 85% or more, 90% or less, 91% or less, 92% or less of the volume of the mold internal space, It may be filled up to 93% or less, 94% or less, or 95% or less. By filling the mold so that it is 60% or more and 95% or less of the volume of the internal space of the mold, the yield when producing a three-dimensional cell structure can be further improved. On the other hand, when the filling rate is less than 60% of the volume of the inner space of the mold, the gap between the contact portions between the cell masses becomes large due to the presence of the cell masses at a low density, and cracks and rough surfaces are formed in the culture step. It is not preferable because it may form a cell culture having the cells. Further, when the filling rate is larger than 95% of the volume of the mold internal space, the physical load due to the contact between the cell clusters increases due to the presence of the cell clusters at an ultra-high density, which is not preferable.
　鋳型内への細胞集団の配置は、１回又は複数回の操作で行うことができる。複数回の場合、１回目の配置工程後、所定の時間が経過した後に、２回目の配置を行う。各回における細胞集団の配置量は同一であっても又は異なっていてもよい。さらに、細胞集団を構成する細胞塊や細胞の種類、又は細胞塊の平均直径も各回で同一であっても又は異なっていてもよい。 The arrangement of the cell population in the template can be performed by one or multiple operations. In the case of a plurality of times, the second placement is performed after a predetermined time has elapsed after the first placement step. The amount of cell population arranged in each round may be the same or different. Furthermore, the cell masses and cell types that make up the cell population, or the average diameter of the cell masses may be the same or different each time.
　鋳型内部空間への配置において、細胞塊や細胞の自然落下による方法、細胞集団流入口から人為的圧力を付与する方法、又は鋳型外部及び／又は内壁部内部から吸引配置する方法等により細胞塊や細胞を積層することが挙げられるが、これらの方法に限られない。ただし、配置に際して細胞に外力が加わると細胞へのダメージを生じ得ることから人為的圧力や吸引力を付与しない自然落下による配置が好ましい。また、本明細書において「積層」とは前記鋳型の内部空間内において複数の前記細胞塊や細胞が重力方向に互いに接触した状態を維持するよう配置することを意味する。 In the arrangement in the mold internal space, the cell mass or the cell mass is arranged by a method of spontaneously dropping the cell mass or cells, a method of applying artificial pressure from the cell population inlet, or a method of suction arrangement from the outside of the template and / or the inside of the inner wall. Stacking cells can be mentioned, but is not limited to these methods. However, if an external force is applied to the cells during placement, damage to the cells may occur, so placement by free fall without applying artificial pressure or suction force is preferable. Further, in the present specification, "stacking" means arranging a plurality of the cell masses and cells in the internal space of the template so as to maintain a state of being in contact with each other in the direction of gravity.
（３）培養工程
　「培養工程」（Ｓ０１０３）は、前記細胞集団配置工程後に鋳型内部空間内に配置した細胞集団を前記鋳型と共に培地中で培養する工程である。「鋳型と共に」とは、鋳型内部空間内に配置された細胞集団をその鋳型ごと培地中に浸漬して培養することをいう。鋳型壁部は通液性のある多孔構造を有するため、培地がその孔を介して流出入することで鋳型内部空間内の細胞集団は培養され得る。内部空間内で各細胞塊及び／又は細胞は、接触を介して互いに接着して鋳型内部空間内で一体化する。その結果、本工程後に鋳型内部に目的の三次元細胞構造体が形成される。
　培地を含む培養槽の形状や大きさは、鋳型が槽内に浸漬可能であれば限定しない。 (3) Culturing Step The “culturing step” (S0103) is a step of culturing a cell population arranged in the template internal space after the cell population arranging step in a medium together with the template. "With a template" means that a cell population arranged in a template internal space is immersed in a medium together with the template and cultured. Since the mold wall portion has a porous structure with liquid permeability, the cell population in the mold internal space can be cultured by the inflow and outflow of the medium through the pores thereof. Within the interior space, the cell masses and / or cells adhere to each other via contact and integrate within the interior space of the mold. As a result, the desired three-dimensional cell structure is formed inside the template after this step.
The shape and size of the culture tank containing the medium are not limited as long as the mold can be immersed in the tank.
　本工程における培養温度、時間、ＣＯ２濃度等の培養条件は特に限定しない。当該分野における常法の範囲で行えばよい。例えば培養温度は下限が２０℃以上、又は３５℃以上、そして上限が４５℃以下、又は４０℃以下であればよいが、好ましくは３７℃である。また、培養期間は、鋳型内部空間内に配置された各細胞塊や細胞が一体化し、目的の三次元細胞構造体が成形される期間とすればよい。限定はしないが、通常は４時間以上、８時間以上、１２時間以上、１６時間以上、２０時間以上、２４時間以上、２日以上、３日以上、４日以上、５日以上、６日以上、７日以上、又は１４日以上、そして２８日未満、２５日以下、２１日以下、又は１８日以下であればよい。好ましくは７日以上２１日以下、より好ましくは１４日以上２１日以下である。培養時のＣＯ２濃度は、４％以上、又は４．５％以上、そして１０％以下、又は５．５％以下であればよいが、好ましくは５％である。良好な培養条件を維持するため、培地を適当な頻度で交換を行うことができる。培地交換の頻度は培養する細胞種、培地の種類、鋳型内部空間の体積や形状によって異なるため、それらの条件を勘案して適宜定めればよい。例えば、５日に１回以上、４日に１回以上、３日に１回以上、２日に１回以上、１日に１回以上で行えばよい。 The culture conditions such as the culture temperature, time, and CO 2 concentration in this step are not particularly limited. It may be done within the scope of the conventional law in the field. For example, the lower limit of the culture temperature may be 20 ° C. or higher or 35 ° C. or higher, and the upper limit may be 45 ° C. or lower or 40 ° C. or lower, but is preferably 37 ° C. In addition, the culture period may be a period during which each cell mass or cell arranged in the template internal space is integrated and a target three-dimensional cell structure is formed. Not limited, but usually 4 hours or more, 8 hours or more, 12 hours or more, 16 hours or more, 20 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more , 7 days or more, or 14 days or more, and less than 28 days, 25 days or less, 21 days or less, or 18 days or less. It is preferably 7 days or more and 21 days or less, and more preferably 14 days or more and 21 days or less. The CO 2 concentration at the time of culturing may be 4% or more, 4.5% or more, and 10% or less, or 5.5% or less, but is preferably 5%. In order to maintain good culture conditions, the medium can be changed at an appropriate frequency. Since the frequency of medium replacement varies depending on the cell type to be cultured, the type of medium, and the volume and shape of the mold internal space, it may be appropriately determined in consideration of these conditions. For example, it may be performed once every 5 days or more, once every 4 days or more, once every 3 days or more, once every 2 days or more, and once a day or more.
　培養中の培地の流動状態は問わない。静置培養でもよいし、流動培養でもよいが、好ましくは流動培養である。 The flow state of the medium during culture does not matter. It may be a static culture or a fluid culture, but it is preferably a fluid culture.
　「静置培養」とは、培養槽内で培地を静置した状態で培養することをいう。「流動培養」とは、培地を流動させる条件下で培養することをいう。鋳型壁部の孔は通液性を有するため、静置培養でも孔を介した培地の自然流出入が可能であるが、培地の流動により、鋳型内部空間内への培地の流出入を能動的に行えることから、流動培養が好ましい。流動培養方法として、例えば、旋回培養法、揺動培養法、又はそれらの組み合わせが挙げられる。 "Standing culture" means culturing the medium in a static state in a culture tank. "Fluid culture" refers to culturing under conditions in which the medium is allowed to flow. Since the pores in the mold wall have liquid permeability, the medium can be naturally flowed in and out through the pores even in static culture, but the flow of the medium actively allows the medium to flow in and out of the mold internal space. Flow culture is preferable because it can be carried out easily. Examples of the flow culture method include a swirl culture method, a swing culture method, or a combination thereof.
　「旋回培養法」（振盪培養法を含む）とは、旋回流による応力（遠心力、求心力）により培養槽内で培地が流動する条件下で培養する方法をいう。具体的には、培養槽を概ね水平面に沿って円、楕円、扁平した円、扁平した楕円等の閉じた軌道を描くように旋回させたり、又は培養槽は静置させたままで攪拌棒や撹拌翼のような撹拌子を用いて槽内の培地を旋回させて培地を流動させたりすることができる。旋回培養法における旋回速度は特に限定されないが、下限は１ｒｐｍ以上、１０ｒｐｍ以上、５０ｒｐｍ以上、６０ｒｐｍ以上、７０ｒｐｍ以上、８０ｒｐｍ以上、８５ｒｐｍ以上、又は９０ｒｐｍ以上とすることができる。一方、上限は２００ｒｐｍ以下、１５０ｒｐｍ以下、１２０ｒｐｍ以下、１１５ｒｐｍ以下、１１０ｒｐｍ以下、１０５ｒｐｍ以下、１００ｒｐｍ以下、９５ｒｐｍ以下、又は９０ｒｐｍ以下とすることができる。旋回培養の際の旋回幅は特に限定されないが、下限は、例えば１ｍｍ以上、１０ｍｍ以上、２０ｍｍ以上、又は２５ｍｍ以上とすることができる。一方、上限は、例えば２００ｍｍ以下、１００ｍｍ以下、５０ｍｍ以下、３０ｍｍ以下、又は２５ｍｍ以下とすることができる。 "Swirl culture method" (including shaking culture method) refers to a method of culturing under conditions in which the medium flows in the culture tank due to stress (centrifugal force, centripetal force) due to the swirling flow. Specifically, the culture tank is swirled so as to draw a closed orbit such as a circle, an ellipse, a flat circle, or a flat ellipse along a horizontal plane, or the culture tank is left to stand and stirred with a stirring rod or agitator. A stirrer such as a wing can be used to swirl the medium in the tank to allow the medium to flow. The swirling speed in the swirling culture method is not particularly limited, but the lower limit may be 1 rpm or more, 10 rpm or more, 50 rpm or more, 60 rpm or more, 70 rpm or more, 80 rpm or more, 85 rpm or more, or 90 rpm or more. On the other hand, the upper limit can be 200 rpm or less, 150 rpm or less, 120 rpm or less, 115 rpm or less, 110 rpm or less, 105 rpm or less, 100 rpm or less, 95 rpm or less, or 90 rpm or less. The swirling width during swirling culture is not particularly limited, but the lower limit can be, for example, 1 mm or more, 10 mm or more, 20 mm or more, or 25 mm or more. On the other hand, the upper limit can be, for example, 200 mm or less, 100 mm or less, 50 mm or less, 30 mm or less, or 25 mm or less.
　「揺動培養法」とは、揺動（ロッキング）撹拌のような直線的な往復運動により培地に揺動流を付与する条件で培養する方法をいう。具体的には、培養槽を概ね水平面に垂直な平面内で揺動させることにより行う。揺動速度は特に限定されないが、例えば１往復を１回とした場合、下限は１分間に２回、４回、６回、８回、又は１０回、一方、上限は１分間に１５回、２０回、２５回、又は５０回で揺動すればよい。揺動の際、垂直面に対して若干の角度、すなわち誘導角度を培養容器につけることが好ましい。揺動角度は特に限定されないが、例えば、下限は０．１°、２°、４°、６°、又は８°、一方、上限は１０°、１２°、１５°、１８°又は２０°とすることができる。
　さらに、上記旋回と揺動とを組み合わせた運動により撹拌しながら培養することもできる。 The "rocking culture method" refers to a method of culturing under conditions in which a rocking flow is applied to the medium by a linear reciprocating motion such as rocking stirring. Specifically, the culture tank is swung in a plane substantially perpendicular to the horizontal plane. The rocking speed is not particularly limited, but for example, when one round trip is once, the lower limit is 2 times, 4 times, 6 times, 8 times, or 10 times per minute, while the upper limit is 15 times per minute. It may swing 20 times, 25 times, or 50 times. When rocking, it is preferable to give the culture vessel a slight angle with respect to the vertical plane, that is, an induction angle. The swing angle is not particularly limited, but for example, the lower limit is 0.1 °, 2 °, 4 °, 6 °, or 8 °, while the upper limit is 10 °, 12 °, 15 °, 18 °, or 20 °. can do.
Furthermore, it is also possible to incubate while stirring by a motion that combines the above-mentioned turning and rocking.
（４）分離工程
　「分離工程」は、前記培養工程後に得られる三次元細胞構造体と前記鋳型とを分離する工程である。本工程は選択的工程である。例えば、鋳型が生体組織と融合し得る素材、例えば、多糖類高分子やゲル化タンパク質で構成される場合、培養工程後の三次元細胞構造体は必ずしも鋳型と分離せず、鋳型と一体の状態で使用することもできる。この場合、本工程は不要である。一方、鋳型が金属やプラスチック等の無生物系素材の場合には、好ましくは培養工程後、少なくとも製造された三次元細胞構造体の使用前には本工程により分離しておくことが望ましい。 (4) Separation Step The “separation step” is a step of separating the three-dimensional cell structure obtained after the culture step and the template. This process is a selective process. For example, when the template is composed of a material that can be fused with living tissue, for example, a polysaccharide polymer or a gelled protein, the three-dimensional cell structure after the culture step does not necessarily separate from the template and is in a state of being integrated with the template. It can also be used in. In this case, this step is unnecessary. On the other hand, when the template is an inanimate material such as metal or plastic, it is desirable to separate the template by this step after the culturing step and at least before using the produced three-dimensional cell structure.
　鋳型から三次元細胞構造体を分離する方法は特に限定はしない。鋳型内部空間と三次元細胞構造体、及び内壁部を有する場合には内壁部と三次元細胞構造体との間に物理的及び／又は化学的な力を付与し、両者を剥離すればよい。物理的な力には、例えば、剪断応力、引張り力、圧力（水圧、空気圧を含む）、温度が含まれる。化学的な力には、例えば、弱いタンパク質分解酵素等の酵素処理が含まれる。物理的な力や化学的な外力が過度に細胞に加わると、細胞死を誘導し得るため外力は弱い方がよい。本工程を経る場合、鋳型内部空間壁面や内壁部表面のような細胞接着面を細胞非接着素材にしておくことで、鋳型内部空間で目的の三次元細胞構造体は成形されるものの、充填された細胞集団は、鋳型内部空間壁面や内壁部表面には接着しないことから、外力をほとんど加えることなく本工程を容易に達成し得る。 The method for separating the three-dimensional cell structure from the template is not particularly limited. If the mold has an internal space, a three-dimensional cell structure, and an inner wall portion, a physical and / or chemical force may be applied between the inner wall portion and the three-dimensional cell structure, and both may be peeled off. Physical forces include, for example, shear stress, tensile force, pressure (including water pressure and air pressure), and temperature. Chemical forces include, for example, enzyme treatments such as weak proteolytic enzymes. When a physical force or a chemical external force is excessively applied to a cell, cell death can be induced, so the external force should be weak. When this step is performed, by making the cell adhesion surface such as the inner wall surface of the mold and the surface of the inner wall a non-cell adhesion material, the target three-dimensional cell structure is formed in the inner space of the mold, but is filled. Since the cell population does not adhere to the inner space wall surface of the mold or the surface of the inner wall portion, this step can be easily achieved with almost no external force applied.
　本工程後に、鋳型から取り出される細胞培養物が本発明の製造方法によって得られる目的の三次元細胞構造体となる。 After this step, the cell culture taken out from the template becomes the target three-dimensional cell structure obtained by the production method of the present invention.
１－４．効果
　本発明の三次元細胞構造体の製造方法によれば、細胞密度が高く、形状均一性と物理的強度が高い三次元細胞構造体を任意の形状で簡便に、かつ安定的に製造することができる。それにより、歩留りが向上し、三次元細胞構造体の大量生産が可能となる。 1-4. Effect According to the method for producing a three-dimensional cell structure of the present invention, a three-dimensional cell structure having high cell density, shape uniformity and physical strength can be easily and stably produced in any shape. Can be done. As a result, the yield is improved and mass production of the three-dimensional cell structure becomes possible.
２．三次元細胞構造体
２－１．概要
　本発明の第２の態様は、三次元細胞構造体である。本発明の三次元細胞構造体は、前記第１態様の三次元細胞構造体の製造方法を用いて得られる。従来法で得られる三次元細胞構造体は、細胞密度が低く形状が不均一になりやすい上に、強度も不十分であった。しかし、前記第１態様の製造方法によれば、所定の平均直径を有する細胞塊を鋳型内部空間内に配置することで細胞を高密度で充填することが可能となり、その後、細胞を鋳型と共に培養することで、細胞密度、形状均一性、及び物理的強度の高い、これまでにない三次元細胞構造体を得ることができる。 2. Three-dimensional cell structure 2-1. Overview A second aspect of the present invention is a three-dimensional cell structure. The three-dimensional cell structure of the present invention can be obtained by using the method for producing a three-dimensional cell structure of the first aspect. The three-dimensional cell structure obtained by the conventional method has a low cell density, tends to have a non-uniform shape, and has insufficient strength. However, according to the production method of the first aspect, the cells can be packed at high density by arranging the cell mass having a predetermined average diameter in the space inside the template, and then the cells are cultured together with the template. By doing so, it is possible to obtain an unprecedented three-dimensional cell structure having high cell density, shape uniformity, and physical strength.
２－２．構造
　本発明の三次元細胞構造体の構造は、前記第１態様の三次元細胞構造体の製造方法に記載の鋳型における内部空間の形状に相同的な形状を有する。それ故に、鋳型によって任意の形状とすることが可能である。本発明の三次元細胞構造体の具体な形状例として、実施例に記載の血管を模した管状構造体の他、耳介のような複雑な形態の構造体が挙げられる。 2-2. Structure The structure of the three-dimensional cell structure of the present invention has a shape homologous to the shape of the internal space in the template according to the method for producing the three-dimensional cell structure of the first aspect. Therefore, it is possible to have an arbitrary shape depending on the mold. Specific examples of the shape of the three-dimensional cell structure of the present invention include a tubular structure that imitates a blood vessel described in Examples, and a structure having a complicated shape such as an auricle.
　構造体を構成する細胞は、細胞集団配置工程で前記鋳型に配置される細胞集団、及び培地中で培養する培養工程によって得られた細胞である。したがって、構造体を構成する細胞の種類は、製造時に配置した細胞集団に含まれる細胞塊や細胞の種類による。また、細胞集団に含まれる個々の細胞塊や細胞は、培養工程で細胞接着によって互いに結合し、一体化した細胞集合体となる。 The cells constituting the structure are the cell population arranged in the template in the cell population arrangement step and the cells obtained by the culture step of culturing in the medium. Therefore, the type of cells constituting the structure depends on the cell mass and the type of cells contained in the cell population arranged at the time of production. In addition, individual cell masses and cells contained in a cell population are bound to each other by cell adhesion in the culture step to form an integrated cell aggregate.
　本発明の三次元細胞構造体は、従来の三次元細胞構造体と比較して細胞密度が顕著に高く、そのため形状均一性と物理的強度の高い特徴を有する。 The three-dimensional cell structure of the present invention has a significantly higher cell density than the conventional three-dimensional cell structure, and therefore has features of high shape uniformity and high physical strength.
２－３．用途
　本発明の三次元細胞構造体は、前記第１態様の製造方法によって得られ、再生医療における移植用部材として使用することができる。すなわち、本発明の三次元細胞構造体によれば、移植用部材のために使用される三次元細胞構造体が提供される。また、本発明の三次元細胞構造体によれば、移植用部材の製造のための三次元細胞構造体の使用が提供される。さらに、本発明の三次元細胞構造体によれば、患者又は被験者に三次元細胞構造体の治療有効量を投与する工程を含む、患者又は被験者に三次元細胞構造体を移植する方法、並びに患者又は被験者の疾患の治療方法が提供される。 2-3. Applications The three-dimensional cell structure of the present invention can be obtained by the production method of the first aspect and can be used as a member for transplantation in regenerative medicine. That is, according to the three-dimensional cell structure of the present invention, a three-dimensional cell structure used for a member for transplantation is provided. Further, according to the three-dimensional cell structure of the present invention, the use of the three-dimensional cell structure for manufacturing a member for transplantation is provided. Further, according to the three-dimensional cell structure of the present invention, a method of transplanting a three-dimensional cell structure into a patient or subject, including a step of administering a therapeutically effective amount of the three-dimensional cell structure to the patient or subject, and a patient. Alternatively, a method of treating the subject's disease is provided.
　移植用部材としては、例えば、国際公開ＷＯ２０１６／０６８２９２号公報や、Ａｎｎａ　Ｄ．Ｄｉｋｉｎａ，ｅｔ　ａｌ．，Ｂｉｏｍａｔｅｒｉａｌｓ，２０１５，５２：４５２－４６２.等において、人工血管形成や軟骨再生に適した移植用部材が知られており、本発明の三次元細胞構造体もそのような用途に使用することができる。 Examples of the transplanting member include International Publication WO2016 / 068292, and Anna D.A. Dikina, et al. , Biomaterials, 2015, 52: 452-462., Etc., known transplanting members suitable for artificial angioplasty and cartilage regeneration, and the three-dimensional cell structure of the present invention can also be used for such applications. it can.
　本明細書における「患者又は被験者」とは、典型的にはヒトであるが、他の動物であってもよい。他の動物としては、例えば、マウス、ラット、ハムスター、モルモット、スナネズミ等のげっ歯類、イヌ、ネコ、ウサギ、ウシ、ウマ、ブタ、ヒツジ、ヤギ、フェレット等の家畜又は愛玩動物、そしてヒト、カニクイザル、アカゲザル、コモンマーモセット、ニホンザル、ゴリラ、チンパンジー等の霊長類が挙げられるが、これらに限定されない。 The "patient or subject" in the present specification is typically a human, but may be another animal. Other animals include, for example, rodents such as mice, rats, hamsters, guinea pigs, snails, livestock or pets such as dogs, cats, rabbits, cows, horses, pigs, sheep, goats, ferrets, and humans. Examples include, but are not limited to, primates such as crab monkeys, red-tailed monkeys, common marmosets, Japanese monkeys, gorillas, and chimpanzees.
２－４．効果
　本発明の三次元細胞構造体は、細胞密度、形状均一性及び物理的強度が高いため、再生医療における移植用部材として利用することができる。 2-4. Effect Since the three-dimensional cell structure of the present invention has high cell density, shape uniformity and physical strength, it can be used as a member for transplantation in regenerative medicine.
　本発明の三次元細胞構造体の製造方法の具体例を以下に示す。ただし、本発明の製造方法は、以下の方法に限定はされない。 A specific example of the method for producing a three-dimensional cell structure of the present invention is shown below. However, the production method of the present invention is not limited to the following methods.
＜方法＞
１．比較例１
（１）脂肪の採取
　インフォームドコンセントを得たドナーＡから以下の方法により脂肪を採取した。予め特定した採取部位（腹部、大腿部等）を１０ｍｍ程切開し、カニューレ（φ２．５／２６０ｍｍ：ＴＰ－２０３、ＭＥＤＩＫＡＮ社）とディスポーザブル注射器（ＴＰ－１０１、ＭＥＤＩＫＡＮ社）を用いて、トゥメッセント液（日本薬局方 生理食塩液１Ｌに日本薬局方 アドレナリン注射液：ボスミン１ｍｇ／１ｍＬを加えた混合液）を皮下組織に注入し、処置をした。処置後、ディスポーザブル注射器（前述）に接続したカニューレ（φ4／２６０ｍｍ：ＴＰ－２０１、φ３／２６０ｍｍ：ＴＰ－２０２、ＭＥＤＩＫＡＮ社）を用いて、脂肪を無菌的に吸引採取した（Ｌｉｐｏｋｉｔ、ＭＥＤＩＣＡＮ社）。その後、前記脂肪を７００×ｇの条件にて５分間遠心分離し、上清を廃液として除去した。 <Method>
1. 1. Comparative Example 1
(1) Collection of fat Fat was collected from donor A who gave informed consent by the following method. A pre-specified collection site (abdomen, thigh, etc.) is incised by about 10 mm, and a cannula (φ2.5 / 260 mm: TP-203, MEDIKAN) and a disposable syringe (TP-101, MEDIKAN) are used to communicate. A solution (a mixture of 1 L of Japanese Pharmacopoeia physiological saline and 1 L of Japanese Pharmacopoeia adrenaline injection: 1 mg / 1 mL of Bosmin) was injected into the subcutaneous tissue for treatment. After the procedure, fat was aseptically sucked and collected using a cannula (φ4 / 260 mm: TP-201, φ3 / 260 mm: TP-202, MEDIKAN) connected to a disposable syringe (described above) (Lipokit, MEDICAN). .. Then, the fat was centrifuged under the condition of 700 × g for 5 minutes, and the supernatant was removed as a waste liquid.
（２）脂肪の酵素処理及び脂肪ＭＳＣの回収
　上述の（１）で取得した脂肪を同量の０．０１％コラゲナーゼを含有するハンクス平衡塩溶液（Ｃａ・Ｍｇ含有）に浸漬し、３７℃で３０分間、２００ｒｐｍの条件にて撹拌することにより脂肪を酵素処理した。処理後に細胞接着が分解され分散状態となった細胞の懸濁液を１０分間、８００×ｇの条件にて遠心分離し、上清を除去した。 (2) Enzyme treatment of fat and recovery of fat MSC The fat obtained in (1) above was immersed in a Hanks balanced salt solution (containing Ca / Mg) containing the same amount of 0.01% collagenase, and at 37 ° C. The fat was enzyme treated by stirring for 30 minutes at 200 rpm. The suspension of cells in which the cell adhesion was decomposed and dispersed after the treatment was centrifuged for 10 minutes under the condition of 800 × g, and the supernatant was removed.
　続いて、終濃度１０％の非働化済みウシ胎児血清（ＦＢＳ）を含むαＭＥＭ（Ａｌｐｈａ　Ｍｏｄｉｆｉｃａｔｉｏｎ　ｏｆ　Ｍｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ　Ｅａｇｌｅ）を加えて懸濁した後、８００×ｇの条件にて５分間遠心分離して、上清を除去した。得られた沈殿物を前記１０％ＦＢＳ含有αＭＥＭにて懸濁し、細胞懸濁液を取得した。この操作を複数回繰り返した。 Subsequently, αMEM (Alpha Modification of Minimum Essential Medium Eagle) containing deactivated fetal bovine serum (FBS) having a final concentration of 10% was added and suspended, and then centrifuged under the condition of 800 × g for 5 minutes. , The supernatant was removed. The obtained precipitate was suspended in αMEM containing 10% FBS to obtain a cell suspension. This operation was repeated multiple times.
　得られた細胞懸濁液から脂肪由来のＭＳＣ（脂肪ＭＳＣ）を分離回収するため、細胞懸濁液に対してフローサイトメーターを用いて、表面抗原ＣＤ９０が陽性を呈する細胞の比率を解析した。ＣＤ９０は、ＭＳＣの代表的な陽性マーカーである。表面抗原解析は、ベクトン・ディッキンソン（ＢＤ）社のＦＡＣＳ　ｃａｎｔｏを用い、解析細胞数：１０，０００ｃｅｌｌｓ、流速設定：Ｍｅｄｉｕｍにて実施した。本測定では、アイソタイプコントロール用抗体として、ＦＩＴＣ Ｍｏｕｓｅ ＩｇＧ１, κ Ｉｓｏｔｙｐｅ Ｃｏｎｔｒｏｌ（ＢＤ社／型番：５５０６１６）を使用した。また、ＣＤ９０抗原に対する抗体としてＦＩＴＣ Ｍｏｕｓｅ Ａｎｔｉ－Ｈｕｍａｎ ＣＤ９０（ＢＤ社／型番：５５５５９５）を使用した。解析の結果、細胞懸濁液から脂肪ＭＳＣが分離できていることを確認した。 In order to separate and recover fat-derived MSCs (fat MSCs) from the obtained cell suspension, the ratio of cells positive for the surface antigen CD90 was analyzed using a flow cytometer with respect to the cell suspension. CD90 is a representative positive marker for MSCs. The surface antigen analysis was carried out using FACS canto of Becton Dickinson (BD), the number of analyzed cells: 10,000 cells, and the flow velocity setting: Medium. In this measurement, FITC Mouse IgG1, κ Isotype Control (BD / model number: 550616) was used as an antibody for isotype control. In addition, FITC Mouse Anti-Human CD90 (BD / model number: 555595) was used as an antibody against the CD90 antigen. As a result of the analysis, it was confirmed that the fat MSC could be separated from the cell suspension.
（３）脂肪ＭＳＣの培養
　上述の（２）で得られた脂肪ＭＳＣを含む細胞集団の一部を、培養容器のＣｅｌｌＳＴＡＣＫ（登録商標）（コーニング社）に４５００個／ｃｍ２の密度で播種し、終濃度にして１０％の非働化済みＦＢＳを含むαＭＥＭにてサブコンフルエントになるまで接着培養した。この接着培養した細胞を、「０継代目の細胞集団」と称する。ＴｒｙｐＬＥ　Ｓｅｌｅｃｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）を用いて０継代目の細胞集団を培養容器から剥離、回収し、１／５量の細胞集団をＣｅｌｌＳＴＡＣＫ（登録商標）（前述）に播種することによって継代培養を行った。この最初の継代培養細胞集団を、「１継代目の細胞集団」と称する。サブコンフルエントに達した時点でＴｒｙｐＬＥ　Ｓｅｌｅｃｔ（前述）を用いて１継代目の細胞集団を剥離し、終濃度にして１０％の非働化済みＦＢＳを含むαＭＥＭを用いて希釈し、遠心分離により回収した。回収した細胞集団に対し、凍結保存液バンバンカー（登録商標）（ＧＣリンフォテック社）を加えて懸濁した後、－８０℃ディープフリーザー下で凍結保存した。その後、解凍して４５００個／ｃｍ２の密度でＣｅｌｌＳＴＡＣＫ（登録商標）（前述）に播種し、サブコンフルエントになるまで継代培養した。このときに継代培養した細胞集団を、「２継代目の細胞集団」と称する。 (3) Culturing of fat MSC A part of the cell population containing the fat MSC obtained in (2) above was seeded in a culture vessel CellSTACK (registered trademark) (Corning Inc.) at a density of 4500 cells / cm 2. The cells were adherently cultured in αMEM containing 10% deactivated FBS at the final concentration until they became subconfluent. These adherently cultured cells are referred to as "0th passage cell population". Subculture by exfoliating and collecting the 0th passage cell population from the culture vessel using TrypLE Select (Thermo Fisher Scientific) and seeding 1/5 amount of the cell population in CellSTACK® (described above). Was done. This first subcultured cell population is referred to as a "first subculture cell population". When the subconfluent was reached, the cell population of the first passage was exfoliated using TrypLE Select (described above), diluted with αMEM containing 10% deactivated FBS at the final concentration, and recovered by centrifugation. .. The collected cell population was suspended by adding a cryopreservation solution Bunbunker (registered trademark) (GC Lymphotech), and then cryopreserved under a -80 ° C deep freezer. Then, it was thawed and seeded in CellSTACK® (described above) at a density of 4500 cells / cm 2, and subcultured until it became subconfluent. The cell population subcultured at this time is referred to as a "second passage cell population".
（４）表面抗原解析
　２継代目の細胞集団対してフローサイトメーターを用いて、各表面抗原（ＣＤ９０の陽性率、ＣＤ７３の陽性率、ＣＤ１０５の陽性率、ＣＤ４５の陽性率及び陰性率）を測定した。表面抗原解析は、ＦＡＣＳ　ｃａｎｔｏ（前述）を用い、解析細胞数：１０，０００ｃｅｌｌｓ、流速設定：Ｍｅｄｉｕｍにて実施した。本測定では、アイソタイプコントロール用抗体として、ＦＩＴＣ Ｍｏｕｓｅ ＩｇＧ１, κ Ｉｓｏｔｙｐｅ Ｃｏｎｔｒｏｌ（前述）、若しくはＰＥ Ｍｏｕｓｅ ＩｇＧ１, κ Ｉｓｏｔｙｐｅ Ｃｏｎｔｒｏｌ（ＢＤ社製／型番：５５０６１７）を使用した。また、ＣＤ９０抗原に対する抗体としてＰＥ ａｎｔｉ－ｈｕｍａｎ ＣＤ９０（Ｔｈｙ１）Ａｎｔｉｂｏｄｙ（ＢｉｏＬｅｇｅｎｄ社製／型番：３２８１１０）を、ＣＤ７３抗原に対する抗体としてＰＥ　Ｍｏｕｓｅ　Ａｎｔｉ－Ｈｕｍａｎ　ＣＤ７３（ＢＤ社製／型番：５５０２５７）を、ＣＤ１０５抗原に対する抗体としてａｎｔｉ－ＣＤ１０５／ＦＩＴＣ（Ａｎｃｅｌｌ社製／型番：３２６－０４０）を、ＣＤ４５抗原に対する抗体としてＦＩＴＣ Ｍｏｕｓｅ Ａｎｔｉ－Ｈｕｍａｎ ＣＤ４５（ＢＤ社製／型番：５５５４８２）を使用した。 (4) Surface antigen analysis Each surface antigen (CD90 positive rate, CD73 positive rate, CD105 positive rate, CD45 positive rate and negative rate) was measured for the cell population of the second passage using a flow cytometer. did. Surface antigen analysis was performed using FACS canto (described above) with the number of cells analyzed: 10,000 cells and the flow velocity setting: Medium. In this measurement, FITC mouse IgG1, κ Mouse Control (described above) or PE mouse IgG1, κ Mouse Control (manufactured by BD / model number: 550617) was used as an antibody for isotype control. Further, PE anti-human CD90 (Th1) Antibody (manufactured by BioLegend / model number: 328110) was used as an antibody against the CD90 antigen, and PE Mouse Anti-Human CD73 (manufactured by BD / model number: 550257) was used as an antibody against the CD73 antigen. Anti-CD105 / FITC (manufactured by Ancell / model number: 326-040) was used as an antibody against the antigen, and FITC Mouse Anti-Human CD45 (manufactured by BD / model number: 555482) was used as an antibody against the CD45 antigen.
　表面抗原解析の結果、２継代目の細胞集団では、ＣＤ９０、ＣＤ７３及びＣＤ１０５の陽性率がいずれも８０％以上であり、ＣＤ４５の陽性率は５％未満（陰性率は９５％以上）であった。以上の結果から、２継代目の細胞集団は、脂肪ＭＳＣを含む細胞集団であることが確認された。 As a result of surface antigen analysis, in the cell population of the second passage, the positive rate of CD90, CD73 and CD105 was 80% or more, and the positive rate of CD45 was less than 5% (negative rate was 95% or more). .. From the above results, it was confirmed that the cell population of the second passage was a cell population containing adipose MSC.
（５）細胞塊の調製
　２継代目の細胞集団を用いて、細胞塊を調製した。まず、終濃度１０％の非働化済みＦＢＳを含むαＭＥＭを細胞培養培地として、２継代目の細胞集団を含む細胞懸濁液を調製した。次に、ＰｒｉｍｅＳｕｒｆａｃｅ（登録商標）プレート９６Ｕに１ウェル当たり６．０×１０４ｃｅｌｌｓとなるように、前記細胞懸濁液を播種した。続いて、播種後のプレートを、５％ＣＯ２下で、３７℃にて２４時間静置培養した。培養後、細胞塊の形成は、顕微鏡（ＯＬＹＭＰＵＳ社製）を用いて確認した。細胞塊の平均直径は、イメージングソフトウェアｃｅｌｌＳｅｎｓｅ（ＯＬＹＭＰＵＳ社製）を用いて顕微鏡像の細胞を撮影した後、得られた撮影像に基づいて任意の３方向から６０個の細胞塊の直径を計測し、算出した。なお、顕微鏡像撮影時の接眼レンズの倍率は１０倍、対物レンズの倍率は４倍とした。 (5) Preparation of cell mass A cell mass was prepared using the cell population of the second passage. First, αMEM containing deactivated FBS having a final concentration of 10% was used as a cell culture medium to prepare a cell suspension containing a second-generation cell population. Next, the cell suspension was seeded on a Prime Surface® plate 96U at 6.0 × 10 4 cells per well. Subsequently, the seeded plate was statically cultured at 37 ° C. for 24 hours under 5% CO 2. After culturing, the formation of cell clusters was confirmed using a microscope (manufactured by OLYMPUS). For the average diameter of the cell mass, after photographing the cells of the microscopic image using the imaging software cellSense (manufactured by OLYMPUS), the diameters of 60 cell masses are measured from any three directions based on the obtained photographed image. , Calculated. The magnification of the eyepiece at the time of photographing the microscope image was 10 times, and the magnification of the objective lens was 4 times.
（６）鋳型の作製
　三次元細胞構造体の製造方法で使用する鋳型を作製した。図２に作製した鋳型（０２００）の概念図を示す。この図で示すように、鋳型は真鍮からなる直径５ｍｍの円柱状底部（０２０１）とポリプロピレン製の多孔メッシュ（サンプラテック株式会社製、カタログ番号：ＷＥＢ１７８１５、孔の長径：２７５．１２μｍ、平均線径：１４０．７４μｍ、開孔率：４３．８％）で構成される略円筒状壁部（０２０２）を有してなる。また鋳型内部空間中心部には、ステンレスからなる直径３ｍｍの円柱状内壁部（０２０３）を有する。作製した鋳型は、円柱状底部（０２０１）、略円筒状壁部（０２０２）及び円柱状内壁部（０２０３）により囲繞された空洞部（０２０４）を有している。鋳型は、シリコン製の土台（図示せず）に固定した。 (6) Preparation of template A template used in the method for producing a three-dimensional cell structure was prepared. FIG. 2 shows a conceptual diagram of the prepared mold (0200). As shown in this figure, the mold is a cylindrical bottom (0201) made of brass with a diameter of 5 mm and a porous mesh made of polypropylene (manufactured by Sampler Tech Co., Ltd., catalog number: WEB17815, hole major axis: 275.12 μm, average wire diameter: It has a substantially cylindrical wall portion (0202) composed of 140.74 μm and an aperture ratio of 43.8%). Further, a cylindrical inner wall portion (0203) having a diameter of 3 mm made of stainless steel is provided in the central portion of the inner space of the mold. The prepared mold has a cylindrical bottom portion (0201), a substantially cylindrical wall portion (0202), and a hollow portion (0204) surrounded by a cylindrical inner wall portion (0203). The mold was fixed to a silicon base (not shown).
（７）三次元細胞構造体の製造
　上述の（５）で得られた細胞塊６０個含む細胞懸濁液を前記鋳型（０２００）に形成された空洞部（０２０４）に流入し、細胞塊を空洞部内にランダムに配置した。その後、配置した細胞塊を前記鋳型と共に細胞培養用培地に浸漬し、５％ＣＯ２下で、３７℃にて２週間静置培養した。なお、細胞培養用培地には、終濃度１０％の非働化済みＦＢＳを含むαＭＥＭを用い、上記培養期間中、２日ごとに培地交換を行った。 (7) Production of Three-Dimensional Cell Structure The cell suspension containing 60 cell masses obtained in (5) above is flowed into the cavity (0204) formed in the template (0200) to form the cell mass. Randomly placed in the cavity. Then, the arranged cell mass was immersed in a cell culture medium together with the template, and statically cultured at 37 ° C. for 2 weeks under 5% CO 2. As the cell culture medium, αMEM containing deactivated FBS having a final concentration of 10% was used, and the medium was exchanged every two days during the above culture period.
（８）鋳型からの三次元細胞構造体の分離
　培養後に空洞部内に形成された三次元細胞構造体を前記鋳型から分離した。分離は、鋳型壁部の多孔メッシュを切断、除去した後、ピンセットを用いて内壁部のステンレス製円柱を三次元細胞構造体から引き抜いて、細胞集団由来の細胞のみで構成された管状三次元細胞構造体を得た。 (8) Separation of three-dimensional cell structure from the template The three-dimensional cell structure formed in the cavity after culturing was separated from the template. For separation, after cutting and removing the porous mesh of the template wall, the stainless steel cylinder of the inner wall is pulled out from the three-dimensional cell structure using tweezers, and tubular three-dimensional cells composed only of cells derived from the cell population. Obtained a structure.
２．実施例１
（１）脂肪の採取
　基本操作は、比較例１の「（１）脂肪の採取」に記載の方法に従った。ただし、本実施例では、インフォームドコンセントを得た比較例１とは異なるドナーＢから脂肪を採取した。
（２）脂肪ＭＳＣの調製
　採取した脂肪の酵素処理及び脂肪ＭＳＣの回収から表面抗原解析までは、比較例１の（２）～（４）に記載の方法に従った。
　なお、表面抗原解析結果は、２継代目の細胞集団のＣＤ９０、ＣＤ７３及びＣＤ１０５の陽性率はいずれも８０％以上であり、ＣＤ４５の陽性率は５％未満（陰性率は９５％以上）であった。この結果から、本実施例の２継代目の細胞集団は、脂肪ＭＳＣを含む細胞集団であることが確認された。
（３）細胞塊の調製
　基本操作は、比較例１の「（５）細胞塊の調製」に記載の方法に従った。ただし、本実施例では、細胞懸濁液播種後のプレートを９６時間で静置培養した。
（４）鋳型の作製
　基本操作は、比較例１の「（６）鋳型の作製」に記載の方法に従った。
（５）三次元細胞構造体の製造
　基本操作は、比較例１の「（７）三次元細胞構造体の製造」に記載の方法に従った。ただし、本実施例では、空洞部内に細胞塊１０９個をランダムに配置した。
（６）鋳型からの三次元細胞構造体の分離
　基本操作は、比較例１の「（８）鋳型からの三次元細胞構造体の分離」に記載の方法に従った。 2. Example 1
(1) Fat collection The basic operation followed the method described in "(1) Fat collection" of Comparative Example 1. However, in this example, fat was collected from donor B, which was different from Comparative Example 1 in which informed consent was obtained.
(2) Preparation of fat MSC From the enzyme treatment of the collected fat and the recovery of the fat MSC to the surface antigen analysis, the methods described in (2) to (4) of Comparative Example 1 were followed.
As a result of surface antigen analysis, the positive rate of CD90, CD73 and CD105 of the second passage cell population was 80% or more, and the positive rate of CD45 was less than 5% (negative rate was 95% or more). It was. From this result, it was confirmed that the cell population of the second passage of this example was a cell population containing adipose MSC.
(3) Preparation of cell mass The basic operation followed the method described in "(5) Preparation of cell mass" of Comparative Example 1. However, in this example, the plate after seeding the cell suspension was statically cultured for 96 hours.
(4) Preparation of Mold The basic operation followed the method described in "(6) Preparation of mold" of Comparative Example 1.
(5) Production of three-dimensional cell structure The basic operation followed the method described in "(7) Production of three-dimensional cell structure" of Comparative Example 1. However, in this example, 109 cell clusters were randomly arranged in the cavity.
(6) Separation of 3D cell structure from template The basic operation followed the method described in "(8) Separation of 3D cell structure from template" of Comparative Example 1.
３．実施例２
（１）脂肪の採取
　基本操作は、比較例１の「（１）脂肪の採取」に記載の方法に従い、比較例１と同じドナーＡから脂肪を採取した。
（２）脂肪ＭＳＣの調製
　採取した脂肪の酵素処理及び脂肪ＭＳＣの回収から表面抗原解析までは、比較例１の（２）～（４）に記載の方法に従った。なお、表面抗原解析では、２継代目の細胞集団におけるＣＤ９０、ＣＤ７３及びＣＤ１０５の陽性率は、いずれも８０％以上であった（具体的にはＣＤ９０：１００％、ＣＤ７３：９８％、ＣＤ１０５：８５％）。また、ＣＤ４５の陽性率は５％未満（陰性率は９５％以上）であった（具体的にはＣＤ４５の陽性率：０％（陰性率：１００％））。以上の結果から、本実施例の２継代目の細胞集団は、脂肪ＭＳＣを含む細胞集団であることが確認された。
（３）細胞塊の調製
　基本操作は、比較例１の「（５）細胞塊の調製」に記載の方法に従った。ただし、本実施例では、細胞懸濁液播種後のプレートの１ウェル当たりに２．０×１０３ｃｅｌｌｓとなるように細胞懸濁液を播種した。
（４）鋳型の作製
　基本操作は、比較例１の「（６）鋳型の作製」に記載の方法に従った。ただし、本実施例では、鋳型の壁部を構成する多孔メッシュの孔の長径を２７５．１２μｍ、平均線径が１４０．７４μｍのものを使用した。この鋳型壁部における多孔メッシュの開孔率は、４３．８％と算出された。
（５）三次元細胞構造体の製造
　基本操作は、比較例１の「（７）三次元細胞構造体の製造」に記載の方法に従った。ただし、本実施例では、前記「（３）細胞塊の調製」で得られた細胞塊１３８個と、比較例１の「（５）細胞塊の調製」で得られた細胞塊１２４個とを混合し、空洞部内にランダムに配置した。
（６）三次元細胞構造体の鋳型からの分離
　基本操作は、比較例１の「（８）鋳型からの三次元細胞構造体の分離」に記載の方法に従った。 3. 3. Example 2
(1) Fat collection In the basic operation, fat was collected from the same donor A as in Comparative Example 1 according to the method described in "(1) Fat collection" of Comparative Example 1.
(2) Preparation of fat MSC From the enzyme treatment of the collected fat and the recovery of the fat MSC to the surface antigen analysis, the methods described in (2) to (4) of Comparative Example 1 were followed. In the surface antigen analysis, the positive rates of CD90, CD73 and CD105 in the second passage cell population were all 80% or more (specifically, CD90: 100%, CD73: 98%, CD105: 85). %). The positive rate of CD45 was less than 5% (negative rate was 95% or more) (specifically, the positive rate of CD45: 0% (negative rate: 100%)). From the above results, it was confirmed that the cell population of the second passage of this example was a cell population containing adipose MSC.
(3) Preparation of cell mass The basic operation followed the method described in "(5) Preparation of cell mass" of Comparative Example 1. However, in this example, the cell suspension was seeded so as to have 2.0 × 10 3 cells per well of the plate after seeding the cell suspension.
(4) Preparation of Mold The basic operation followed the method described in "(6) Preparation of mold" of Comparative Example 1. However, in this example, the holes of the porous mesh constituting the wall portion of the mold had a major axis of 275.12 μm and an average wire diameter of 140.74 μm. The aperture ratio of the porous mesh in the mold wall was calculated to be 43.8%.
(5) Production of three-dimensional cell structure The basic operation followed the method described in "(7) Production of three-dimensional cell structure" of Comparative Example 1. However, in this example, 138 cell masses obtained in the above-mentioned "(3) Preparation of cell mass" and 124 cell masses obtained in "(5) Preparation of cell mass" of Comparative Example 1 are used. They were mixed and randomly placed in the cavity.
(6) Separation of 3D cell structure from template The basic operation followed the method described in "(8) Separation of 3D cell structure from template" of Comparative Example 1.
＜結果＞
　比較例１、実施例１及び実施例２の結果について、以下の表１に示す。 <Result>
The results of Comparative Example 1, Example 1 and Example 2 are shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表中、「細胞培養物の亀裂」において、○は亀裂が確認できた場合を、×は確認できなかった場合を示す。また、「鋳型と三次元細胞構造体との分離容易性」において、○は分離が容易だった場合を、×は分離が困難であった場合を示す。さらに、「目的三次元細胞構造体の取得」において、○は取得ができた場合を、×はできなかった場合を示す。そして、「三次元細胞構造体の均一性」において、○は均一であった場合を、×は不均一であった場合を示す。 In the table, in the "crack of cell culture", ○ indicates the case where the crack was confirmed, and × indicates the case where the crack could not be confirmed. Further, in the "ease of separation between the template and the three-dimensional cell structure", ◯ indicates a case where the separation was easy, and × indicates a case where the separation was difficult. Further, in the "acquisition of the target three-dimensional cell structure", ◯ indicates the case where the acquisition was possible, and × indicates the case where the acquisition was not possible. Then, in "homogeneity of the three-dimensional cell structure", ◯ indicates a case where it was uniform, and × indicates a case where it was non-uniform.
　表１に示すように、実施例１における細胞集団中の所望径（平均直径が５０μｍ以上、７５０μｍ以下）の細胞塊の比率（所望径細胞塊含有率）は１００％であった。また、実施例２における所望径細胞塊含有率は５３％であった。さらに、比較例１における所望径細胞塊含有率は０％であった。 As shown in Table 1, the ratio (desired diameter cell mass content) of cell clusters having a desired diameter (average diameter of 50 μm or more and 750 μm or less) in the cell population in Example 1 was 100%. The desired diameter cell mass content in Example 2 was 53%. Furthermore, the desired diameter cell mass content in Comparative Example 1 was 0%.
　表１に示すように、実施例１及び２では、細胞塊を含む細胞集団を鋳型内部空間の体積の６０％以上まで充填することができた（すなわち、細胞塊充填率が６０％以上であった）が、比較例１では細胞塊をそのような高密度で充填することができなかった（すなわち、細胞塊充填率が６０％未満であった）。 As shown in Table 1, in Examples 1 and 2, the cell population containing the cell mass could be filled up to 60% or more of the volume of the template internal space (that is, the cell mass filling rate was 60% or more). However, in Comparative Example 1, the cell mass could not be filled at such a high density (that is, the cell mass filling rate was less than 60%).
　また、表１、図３及び図４に示すように、実施例１及び２では、培養工程において細胞塊同士が高密度の状態で均一に融合し、目視可能な亀裂はなく、滑らかな表面を有する細胞培養物として形成された。一方、表１及び図５に示すように、比較例１では、培養工程において細胞塊同士が不均一に融合した結果、目視可能な亀裂を有し、かつ粗い表面を有する細胞培養物が形成された。 Further, as shown in Tables 1, 3 and 4, in Examples 1 and 2, the cell clusters were uniformly fused in a high-density state in the culture step, and there were no visible cracks and a smooth surface was obtained. Formed as a cell culture with. On the other hand, as shown in Table 1 and FIG. 5, in Comparative Example 1, as a result of non-uniform fusion of cell masses in the culture step, a cell culture having visible cracks and a rough surface was formed. It was.
　さらに、表１、図６及び図７に示すように、実施例１及び２では、特に大きな力を加えることなく鋳型内部空間と内壁部で形成される空洞部から三次元細胞構造体を取り出すことができた。特に、三次元細胞構造体をステンレス製円柱（鋳型内部空間の内壁部）から抜去した際、ピンセットを用いて三次元細胞構造体を掴むことができたため、三次元細胞構造体をステンレス製円柱から容易に分離することができた。一方、表１及び図８に示すように、比較例１では、三次元細胞構造体をステンレス製円柱から抜去しようと試みたものの、三次元細胞構造体の表面に多数の亀裂が見られ、その物理的強度が著しく不足していたため、原形を維持したまま三次元細胞構造体をステンレス製円柱から分離することはできなかった。 Further, as shown in Tables 1, 6 and 7, in Examples 1 and 2, the three-dimensional cell structure is taken out from the cavity formed by the mold internal space and the inner wall portion without applying a particularly large force. Was made. In particular, when the three-dimensional cell structure was removed from the stainless steel cylinder (the inner wall of the mold internal space), the three-dimensional cell structure could be grasped using tweezers, so that the three-dimensional cell structure was removed from the stainless steel cylinder. It could be easily separated. On the other hand, as shown in Table 1 and FIG. 8, in Comparative Example 1, although an attempt was made to remove the three-dimensional cell structure from the stainless steel cylinder, a large number of cracks were found on the surface of the three-dimensional cell structure. Due to the significant lack of physical strength, it was not possible to separate the 3D cell structure from the stainless steel cylinder while maintaining its original shape.
　加えて、表１及び図７に示すように、実施例１及び２では、鋳型から分離した後も三次元細胞構造体は原形を維持しており、所望の管状の三次元細胞構造体を取得することができた。一方、比較例１では、三次元細胞構造体が著しく脆く、所望の形状を有する三次元細胞構造体を取得することはできなかった。 In addition, as shown in Tables 1 and 7, in Examples 1 and 2, the three-dimensional cell structure maintained its original shape even after separation from the template, and a desired tubular three-dimensional cell structure was obtained. We were able to. On the other hand, in Comparative Example 1, the three-dimensional cell structure was extremely brittle, and it was not possible to obtain a three-dimensional cell structure having a desired shape.
　以上より、本発明の製造方法により、物理的強度が高く、均一な形状の三次元細胞構造体を効率よく製造することができた。さらに、上記製造方法により得られた三次元細胞構造体は、物理的強度が高く、均一な形状であった。
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 From the above, it was possible to efficiently produce a three-dimensional cell structure having high physical strength and a uniform shape by the production method of the present invention. Further, the three-dimensional cell structure obtained by the above-mentioned production method had high physical strength and a uniform shape.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (14)

  1. 　三次元細胞構造体の製造方法であって、
    　平均直径が５０μｍ以上、７５０μｍ以下の細胞塊を含む細胞集団を、鋳型の内部空間内に配置する細胞集団配置工程、及び
    　前記細胞集団を前記鋳型と共に培地中で培養する培養工程
    を含み、
    　前記鋳型の壁部が通液可能な多孔構造を有する、前記製造方法。     It is a method for manufacturing a three-dimensional cell structure.
    It comprises a cell population placement step of arranging a cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less in the inner space of a template, and a culture step of culturing the cell population together with the template in a medium.
    The production method, wherein the wall portion of the mold has a porous structure through which liquid can pass.
  2. 　前記細胞集団が前記細胞塊を５０％以上含む、請求項１に記載の製造方法。 The production method according to claim 1, wherein the cell population contains 50% or more of the cell mass.
  3. 　前記鋳型の内部空間が円筒状である、請求項１又は２に記載の製造方法。 The manufacturing method according to claim 1 or 2, wherein the internal space of the mold is cylindrical.
  4. 　前記細胞塊の平均直径（μｍ）に対する前記多孔構造における孔の長径（μｍ）及び／又は最大対角線（μｍ）との比率が１．０より大きく、３．０以下である、請求項１～３のいずれか一項に記載の製造方法。 Claims 1 to 3 in which the ratio of the major axis (μm) and / or the maximum diagonal line (μm) of the pores in the porous structure to the average diameter (μm) of the cell mass is greater than 1.0 and less than or equal to 3.0. The manufacturing method according to any one of the above.
  5. 　前記多孔構造における開孔率が４０％以上、９０％以下である、請求項１～４のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 4, wherein the pore opening ratio in the porous structure is 40% or more and 90% or less.
  6. 　前記細胞集団配置工程において、前記細胞塊を含む細胞集団を前記鋳型内部空間の体積の６０％以上、９５％以下まで充填する、請求項１～５のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 5, wherein in the cell population arrangement step, the cell population containing the cell mass is filled to 60% or more and 95% or less of the volume of the mold internal space.
  7. 　前記細胞集団配置工程において、前記鋳型に人為的圧力を付与することなく前記細胞塊を積層する、請求項１～６のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 6, wherein in the cell population placement step, the cell clusters are laminated without applying artificial pressure to the template.
  8. 　前記培養工程における培養期間が４時間以上、２８日未満である、請求項１～７のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 7, wherein the culture period in the culture step is 4 hours or more and less than 28 days.
  9. 　前記培養工程後に得られる三次元細胞構造体と前記鋳型とを分離する分離工程をさらに含む、請求項１～８のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 8, further comprising a separation step of separating the three-dimensional cell structure obtained after the culture step and the template.
  10. 　前記鋳型の細胞接触面が細胞非接着素材で構成されている、請求項９に記載の製造方法。 The production method according to claim 9, wherein the cell contact surface of the template is made of a non-cell adhesive material.
  11. 　前記細胞集団配置工程に先立ち、平均直径が５０μｍ以上、７５０μｍ以下の細胞塊を含む細胞集団を調製する細胞集団調製工程をさらに含む、請求項１～１０のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 10, further comprising a cell population preparation step of preparing a cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less prior to the cell population arrangement step.
  12. 　前記細胞塊を構成する細胞が、間葉系幹細胞、線維芽細胞、内皮細胞及び軟骨細胞からなる群より少なくとも一つ選択される、請求項１～１１のいずれか一項に記載の製造方法。 The production method according to any one of claims 1 to 11, wherein at least one cell constituting the cell mass is selected from the group consisting of mesenchymal stem cells, fibroblasts, endothelial cells and chondrocytes.
  13. 　前記間葉系幹細胞が、骨髄、脂肪、胎児付属物又は歯髄に由来するものである、請求項１２に記載の製造方法。 The production method according to claim 12, wherein the mesenchymal stem cells are derived from bone marrow, fat, fetal appendages or dental pulp.
  14. 　三次元細胞構造体であって、
    　平均直径が５０μｍ以上、７５０μｍ以下の細胞塊を含む細胞集団を、鋳型の内部空間内に配置した後、前記細胞集団を前記鋳型と共に培地中で培養して得られ、前記鋳型の壁部が通液可能な多孔構造を有する、前記三次元細胞構造体。     It is a three-dimensional cell structure
    A cell population containing a cell mass having an average diameter of 50 μm or more and 750 μm or less is placed in the inner space of the template, and then the cell population is cultured in a medium together with the template. The three-dimensional cell structure having a liquid-possible porous structure.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006075191A (en) * 2004-09-07 2006-03-23 Olympus Corp Artificial blood vessel, its production method and production device
JP2016000029A (en) * 2014-05-20 2016-01-07 国立大学法人佐賀大学 Tissue body forming apparatus and tissue body forming kit
CN110055174A (en) * 2019-02-27 2019-07-26 中国医学科学院生物医学工程研究所 A kind of classification inoculation apparatus and its application method for three-dimensional cell cultivation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006075191A (en) * 2004-09-07 2006-03-23 Olympus Corp Artificial blood vessel, its production method and production device
JP2016000029A (en) * 2014-05-20 2016-01-07 国立大学法人佐賀大学 Tissue body forming apparatus and tissue body forming kit
CN110055174A (en) * 2019-02-27 2019-07-26 中国医学科学院生物医学工程研究所 A kind of classification inoculation apparatus and its application method for three-dimensional cell cultivation

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